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Guo Guo Guo-qing Song CSS/HRT 451 CSS/HRT 451 CSS/HRT 451 Biolistic-mediated transformation f Gene Delivery System y Agrobacterium y Viral vectors y Biolistic y Microinjection y PEG - Polyethylene Glycol y Electroporation BiolisticBiolistic TransformationTransformation ------Advantage and Disadvantage f Advantage:

y This method can be use to transform all plant species. y No binary vector is required. y Transformation protocol is relatively simple. f Disadvantage:

y Difficulty in obtaining single copy transgenic events. y High cost of the equipment and microcarriers. y Intracellular target is random (cytoplasm, nucleus, vacuole, plastid, etc.). y Transfer DNA is not protected. Gene Delivery System ------Biolistic-mediated transformation

Known as:

Particle Bombardment Biolistics Microprojectile bombardment Particle acceleration Particle inflow gun Gene gun

Using a gene gun directly shoots a piece of DNA into the recipient plant tissue.

Tungsten or gold beads are coated in the gene of interest and fired through a stopping screen, accelerated by Helium, into the plant tissue. The particles pass through the plant cells, leaving the DNA inside. Biolistic-Mediated Gene Transfer ------Mechanism Biolistic-Mediated Gene Transfer ------Equipment

PDS-1000/He The Helios Gene Gun www.bio-rad.com/genetransfer/ Particle Inflow Guns (PIG) http://www.oardc.ohio-state.edu/plantranslab/PIG.htm Biolistic-Mediated Gene Transfer ------Equipment- PDS-1000/He

PDS-1000/He

DNA-coated microcarriers are loaded on microcarrier. Micro-carriers are shot towards target tissues during helium gas decompression. A stopping screen placed allowing the coated microprojectiles to pass through and reach the target www.bio-rad.com/genetransfer/ cells. Biolistic-Mediated Gene Transfer ------PDS-1000/He References

Arnold D et al., Proc Natl Acad Sci USA 91, 9970–9974 (1994) Castillo AM et al., Biotechnology 12, 1366–1371 (1994) Duchesne LC et al., Can J For Res 23, 312–316 (1993) Fitzpatrick-McElligott S, Biotechnology 10, 1036–1040 (1992) Hartman CL et al., Biotechnology 12, 919–923 (1994) Heiser WC, Anal Biochem 217, 185–196 (1994) Lo DC et al., Neuron 13, 1263–1268 (1994) Sanford JC et al., Technique 3, 3–16 (1991) Shark KB et al., 480–485 (1991) Smith FD et al., J Gen Microbiol 138, 239–248 (1992) Svab Z and Maliga P, Proc Natl Acad Sci USA 90, 913–917 (1993) Toffaletti DL et al., J Bacteriol 175, 1405–1411 (1993) www.bio-rad.com/genetransfer/ Biolistic-Mediated Gene Transfer ------Equipment- Particle Inflow Gun

Finer JJ, P Vain, MW Jones, MD McMullen (1992) Development of the particle inflow gun for DNA delivery to plant cells. Plant Cell Reports 11:232-238. Vain P, N Keen, J Murillo, C Rathus, C Nemes, JJ Finer (1993) Development of the Particle Inflow Gun. Plant Cell Tiss Org Cult 33:237-246. Biolistic-Mediated Gene Transfer ------Equipment- Helios Gene Gun www.bio-rad.com/genetransfer/

f The helium pulse sweeps the DNA- or RNA-coated gold microcarriers from the inside wall of the sample cartridge. f The microcarriers accelerate for maximum penetration as they move through the barrel, while the helium pulse diffuses outward. f The spacer maintains the optimal target distance for in vivo applications and vents the helium gas away from the target to minimize cell surface impact. Biolistic-Mediated Gene Transfer ------Helios Gene Gun System References

Fynan EF et al., DNA vaccines: Protective immunizations by parenteral, mucosal, and gene-gun inoculations, Proc Natl Acad Sci USA 90, 11478–11482 (1993)

Qiu P et al., Gene gun delivery of mRNA in situ results in efficient transgene expression and genetic immunization, Gene Ther 3, 262– 268 (1996)

Sun WH et al., In vivo cytokine gene transfer by gene gun reduces tumor growth in mice, Proc Natl Acad Sci USA 92, 2889–2893 (1995)

Sundaram P et al., Particle-mediated delivery of recombinant expression vectors to rabbit skin induces high-titered polyclonal antisera (and circumvents purification of a protein immunogen), Nucleic Acids Res 24, 1375–1377 (1996)

Tang DC et al., Genetic immunization is a simple method for eliciting an immune response, Nature 356, 152–154 (1992) www.bio-rad.com/genetransfer/

BiolisticBiolisticTransformation Transformation ------Parameters

A number of parameters has been identified and need to be considered carefully in experiments involving particle bombardment

Parameter categories:

- Physical parameters - Biological parameters - Environmental parameters BiolisticBiolistic TransformationTransformation ------Parameters -Physicalparameters f Nature, chemical and physical properties of the metal particles used as a macrocarrier for the foreign DNA Î Particles should be high enough mass in order to possess adequate momentum to penetrate into appropriate tissue. Î Suitable metal particles include gold, tungsten, palladium, rhodium, platinum and iridium. Î Metals should be chemically inert to prevent adverse reaction with DNA and cell components. Î Additional desirable properties for the metal include size and shape, as well as agglomeration and dispersion properties—diameter 0.36-6 μm. f Nature, preparation and binding of DNA onto the particles fTarget tissue -Biologicalparameters - Environmental parameters BiolisticBiolistic TransformationTransformation ------Parameters -Physicalparameters f Nature, chemical and physical properties of the metal particles used as a macrocarrier for the foreign DNA f Nature, preparation and binding of DNA onto the particles

Î The nature of DNA (single vs double stranded, circular vs linerized DNA). Optimal: double stranded circular DNA molecules (e.g. plasmid) Î In the process of coating the metal particls with DNA certain additives

such as spermididne and CaCl2 appear to be useful.

fTarget tissue

-Biologicalparameters - Environmental parameters BiolisticBiolistic TransformationTransformation ------Parameters -Physicalparameters f Nature, chemical and physical properties of the metal particles used as a macrocarrier for the foreign DNA f Nature, preparation and binding of DNA onto the particles fTarget tissue Î It is important to target the appropriate cells that are competent for both transformation and regeneration.

Î Depth of penetration is one of the most important variables in order to achieve particle delivery to particular cells.

-Biologicalparameters - Environmental parameters BiolisticBiolistic TransformationTransformation ------Parameters -Physicalparameters -Biologicalparameters

fTemperature, photoperiod and humidity

Î These parameters have a direct effect on the physiology of tissues.

Î Such factors will influence receptiveness of target tissue to foreign DNA delivery and also affect its susceptibility to damage and injury that may adversely affect the outcome of transformation process. Î Some explants may require a “healing” period after bombardment under special regiments of light, temperature, and humidity.

- Environmental parameters BiolisticBiolistic TransformationTransformation ------Parameters -Physicalparameters -Biologicalparameters - Environmental parameters

Î Nature of explants as well as pre- and post-bombardment culture conditions. Î Explants derived from plants that are under stress will provide inferior materials for bombardment experiments. Î Metal particles need to be directed to the nucleus. Î Transformation frequencies may also be influenced by cell cycle stage.

Î Osmotic pretreatment of target tissues has also been shown to be of importance. Î Physical trauma and tungsten toxicity were found to reduce efficiency of transformation in experiments performed with tobacco cell suspension culture. BiolisticBiolistic TransformationTransformation ------Summary

For biolistic transformation, tungsten or gold particles are coated with DNA and accelerated towards target plant tissues. Most devices use compressed helium as the force to accelerate the particles.

The particles punch holes in the plant cell and ususally petetrate only 1-2 cell layerswall . Particle bombardment is a physical method for DNA introduction.

The DNA-coated particles can end up either near or in the nucleus,where the DNA comes off the particles and integrated into plant chromosomal DNA.

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Subject Name Botany

Paper Name Plant Genetic Engineering

Module Name/Title Direct gene transfer strategies

Module Id Pre-requisites Basic knowledge about plant genetic engineering Objectives To create awareness instudents about different strategies for direct gene transfer Keywords Genetic engineering, restriction enzymes, ligases, phosphatises

Structure of Module / Syllabus of a module (Define Topic / Sub-topic of module)

Direct gene transfer strategies

2. 2. Development Team

Role Name Affiliation

Subject Coordinator Dr. Sujata Bhargava Savitribai Phule Pune University

Paper Coordinator Dr. Rohini Sreevathsa National Research Centre on Plant Biotechnology, Pusa, New Delhi Content Writer/Author (CW) Dr. Rohini Sreevathsa Content Reviewer (CR) Dr.Rohini Sreevathsa Language Editor (LE) Dr.Rohini Sreevathsa

TABLE OF CONTENTS 1. Introduction 2. Biolistics 3. Electroporation 4. Microinjection 5. Liposome mediated gene transfer /Lipofection 6. Polyethylene glycol (PEG) mediated gene transfer 7. Silicon carbide fiber-mediated DNA delivery 8. Direct DNA uptake by mature zygotic embryos 9. Summary and conclusion

Introduction Methods for introduction of foreign genes into plants without the involvement of vectors like Agrobacterium tumefaciensare called ‘direct gene transfer methods’. The methods involve transfer of small amounts of DNA into the plant cell when it is transiently permeabilized. Various types of direct gene transfer strategies are being followed by scientists. Earlier, the direct gene transfer methodologies were used to transform monocots which were less amenable to Agrobacteriumtransformation. In this module we shall study various gene transfer methods used to transfer naked DNA into plant protoplasts, cells or tissues of an intact plant.

Particle gun (Biolistics) Biolistics is an abbreviation of biological ballistics. The particle or gene gun is called a “biolistic particle delivery system.”Thebiolisticsmethod is themost effective method among the various direct gene transfer methods. In this tungsten or gold particles are coated with DNA to be transformed and bombarded into plant tissue. The Biolistic PDS-1000/He instrument uses pressurized helium to accelerate sub-cellular sized microprojectiles coated with DNA (or other biological material) over a range of velocities necessary to optimally transform many different cell types. The system consists of abombardment chamber (main unit)and connecting tube for attachment to vacuum source, and all components necessary for attachment and delivery of high pressure helium to the main unit (helium regulator, solenoid valve, and connecting tube)

Figure 1. Biolisticsapparatus setup instrument

The Biolistics process The Biolistic PDS-1000/He system(Fig.2) uses high pressure helium, released by a rupture disk, and partial vacuum to propel a macrocarrier sheet loaded with millions of microscopic tungsten or gold microcarriers toward target cells at high velocity. The microcarriers are coated with DNA or other biological material for transformation. The macrocarrier is halted after a short distance by a stopping screen. The DNA-coated microcarriers continue traveling toward the target to penetrate and transform the cells. The launch velocity of microcarriers for each bombardment is dependent upon pressure of helium (rupture disk selection), amount of vacuum in the bombardment chamber, distance from the rupture disk to macrocarrier (A), macrocarrier travel distance to stopping screen (B), and distance between stopping screen and target cells (C). The original Biolistic device used a gunpowder explosion to accelerate DNA-coated microcarriers into target cells. The helium technology used in the current PDS-1000/He system has primary advantages of providing cleaner, safer, and more

reproducible particle acceleration. This stems from the use of rupture disks that burst at a defined pressure. In addition, helium causes less tissue damage.

Figure 2. Representation ofBiolistics process

There are various factors which affect the transformation efficiency using this methodology as it depends on the tissue to be transformed, the gas pressure; the path of the microprojectiles etc. one of the major disadvantage of the methodology is the expensive particle gun that is used for the transformation.

Electroporation Introduction of plasmid DNA into cells by exposing them to brief voltage pulses resulting in development of transient pores in the plasmalemma is called electroporation. The experiment is carried out under two conditions involving (a) low voltage-long pulses (300- 400 V cm-1 for 10-50 ms) and (b) high voltage and short pulses 1000-1500 V cm-1 for 10 µs). Generally, low voltage long pulses produces high rates on transient expression whereas, high voltage short pulses give rise to stable integration. The DNA that is electroporated migrates into the pores on the plasmalemma and integrates into the genome. Electroporation has been successfully used to transform all the major cereals particularly rice. Initially, protoplasts were used as explants but later on other tissues could also be used as explants for transformation. However, it involves the treatment of the explants before

and after electroporation with high osmotic buffers. The efficiency of the transformation depends on the type of tissue used, treatment with the high osmotic buffers and the electroporation conditions. Some studies have shown that linear rather than circularized plasmid DNA and addition of spermidine to the incubation buffers also has an influence on transformation efficiency. The method has also been used to generate transformants in a wide variety of species. One major advantage of the methodology is that it retains the tissues in the same physiological state after the electroporation as they were before transformation.

Microinjection Thisdirect gene transfer methodology involves injection of solubilizedDNA using capillary glass micropipettes with the help of micromanipulators. Protoplasts are the preferred explants for transformation because cell wall interferes with the microinjection. The methodology is cumbersome and time consuming because 40-50 protoplasts canonly be injected in an hour. To adopt this procedure, it is better to take cells with dense cytoplasm with smaller vacuoles because larger vacuoles hinder the delivery of DNA into the nucleus.

Liposome mediated gene transfer/Lipofection Liposome mediated gene transfer or Lipofection is a very efficient technique used to transfer genes in bacterial, animal and plant cells. Liposomes have been explored as a delivery system for DNA as early as in 1979. Liposomes are synthetic analogues of the phospholipid bilayer of the cellular membraneandcan act as delivery agents. These compounds contain phospholipids, hydrophobic and hydrophilic regions which allow for the formation of circular lipid molecules under aqueous conditions help to carry nucleic acids. In the presence of DNA, liposomes encapsulate the DNA fragments and later adhere to the cell membranes and fuse with them to transfer DNA fragments, thereby creating an efficient delivery system (Fig. 3).

Figure 3. Schematic representation of the liposome-mediated DNA delivery

Advantages 1. Very easy to perform 2. Long term stability. 3. Low toxicity. 4. Protection of nucleic acid from degradation. Disadvantages 1. Relatively low transduction efficiency

Polyethylene glycol (PEG) mediated gene transfer/Direct Gene Uptake by Protoplasts It is the oldest reliable method of direct gene transfer. This method has been very useful and applied to several plant species. This method is utilized for protoplast only. Protoplasts are cells without rigid cellulose walls. The plant protoplasts treated with PEG and divalent

cations destabilize the plasma membrane of the plant protoplast and render it permeable to naked DNA. Thus DNA enters the nucleus and integrates into the host genome.

Advantages 1. Less toxic 2. Inexpensive supplies and equipment 3. More resistant to nonspecific protein adsorption 4. Readily adapted to a wide range of plant species and tissues. Disadvantages 1. Tedious procedure 2. Regeneration of fertile plants from protoplasts is problematic for some species. 3. The DNA used for transformation is vulnerable to degradation and rearrangement.

Silicon carbide fibre-mediated DNA delivery This method does not involve any equipment but involves the mixing of the cells with DNA and the fibres in a buffer solution and vortexed. The fibres which are about 0.3-0.6 µm in diameter and 10-100 µm long penetrate the plasma membrane allowing the DNA to gain access into the cell. The strategy has drawbacks with respect to the plant material used for transformation and the use of fibres which require careful handling.

Direct DNA uptake by mature zygotic embryos The principle of the methodology is that when imbibed in a solution containing the plasmid DNA, isolated dry embryos take up the DNA and the T-DNA is integrated in the genome. The strategy has been utilized in wheat, rye, barley, pea and bean. Dry seeds whose coats are removed and imbibed also take up the DNA. The integration of the T-DNA was observed in wheat by PCR analysis. However, studies need to be carried out for understanding the pattern of DNA entry and integration into the genome.

Summary and conclusions Several methods of DNA delivery to plant cells besides Agrobacterium mediated transformation are available.These methods transfer naked plasmid DNA carrying gene of interest and selectable marker gene directly into plant cells. Integration is uncertain, but is

seen to occur and leads to successful transformation. Biolistic transfer has an advantage over other methods of direct gene transfer because this method can be used with intact plant tissues. Direct DNA delivery methods using protoplasts are useful to study transient gene expression. The protoplasts can give stable transformants if method for regeneration is available.The biolistic DNA transfer methods is widely used for getting transformants, especially of recalcitrant plants, but have a disadvantage that entry of multiple copies of the plasmid may cause gene silencing. Table 1. Comparison between Agrobacterium mediated transformation method, and two direct DNA transfer methods.

Further reading 1. Kohli, A., Leech, M., Vain, P., Laurie D. A., Christou, P. (1998) Transgene organization in rice engineered through direct gene transfer supports a two phase integration mechanism mediated by the establishment of integration hot spots. PNAS USA 95, 7203- 7208. 2. Sorokin, A. P., Ke, X. Y., Chen D. F., Elliot, M. C. (2000) Production of fertile transgenic wheat plants via tissue electroporation. Plant Science 156, 227-233. 3. Do cite Kikkert JR, Vidal JR &Reisch BI (2004) article no. 4 from “ Methods in Molecular Biology” vol. 286. 4. Article “What is biolistics” by Dr Padma kumar(2010)

Books

1. Biotechnology: Expanding Horizons (2010) by B D Singh. 2. Plant Tissue Culture, Development, and Biotechnology (2010) by Robert N. Trigiano and Dennis J. Gray 3. Plant Biotechnology: genetic manipulation of plants (2008) by Adrian Slater, Nigel Scott and Mark Fowler 4. Introduction to Plant Biotechnology (2009) by H S Chawla

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Module Name/Title < Host genes in entry and integration of T-DNA> Module Id Pre-requisites Basic knowledge Agrobacterium mediated transformation Objectives To make the students aware of the role played by host gene products in entry and integration of T-DNA. Keywords T-DNA integration, host genome, rat mutants, hat mutants, VIP1, VIP2

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, and integration of T- DNA> < Host genes in entry , and integration of T- DNA>

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Subject Coordinator Savitribai Phule Pune University Paper Coordinator National Research Centre on Plant Biotechnology, Pusa, New Delhi Content Writer/Author (CW)

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TABLE OF CONTENTS (for textual content) Introduction Entry of T-DNA into cytoplasm of plant cell Transport of T-DNA into nucleus Integration of T-DNA Regulation of expression of T-DNA Summary and conclusions References

Introduction T-DNA is a segment of DNA present on the Ti plasmid of Agrobacterium tumefaciens. It has 24-bp repeats at the left and right borders, which are recognised by the virD1/D2 endonuclease, and a single stranded T-DNA molecule is excised and replaced by strand replacement synthesis. A close contact between Agrobacterium and the host plant cell is necessary for the transfer of T-DNA. Numerous bacterial genes are responsible for the initial attachment. Type 4 secretory system (T4SS) components, virB1, virB2 and virB5 interact with plant receptors and the other proteins coded by the vir B operon, along with virD4 proteins form a pore through which T-DNA and other proteins are transferred from the bacterium to plant cell. In this module we will study the role of host proteins involved in T-DNA transfer between Agrobacterium and host cells and its integration into the plant genome.

Entry of T-DNA into cytoplasm of plant cell For T-DNA transport, attachment of the Agrobacterium to plant cell is essential. Bacterial glucans and cellulose fibrils are involved in the formation of biofilm in which the bacteria are embedded at the plant surface. A plant cellulose synthase homolog CSLA9 is thought to modify the plant cell surface, thus facilitating bacterial attachment. The T4SS formed by bacterial virB proteins traverses the plant cell wall and plant cell membrane to inject the T-DNA and other macromolecules called effector proteins into the host cell cytoplasm,

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with the T-pilus acting as a hollow needle (Kado, 2000). It is also proposed that T-DNA are transferred through a protein channel generated in the lipid membrane by bacterial virE gene (Dumas et al., 2001).Some plant proteins have been shown to interact with the T-pilus, namelyBTI1, 2, and 3, which are related proteins of unknown function, and a fourth protein, which is a membrane-associated GTPase, AtRAB8. These proteins are thought to play the role of plant receptors mediating interaction with the T-pilus,and susceptibility to Agrobacterium correlates with their expression levels (Hwang and Gelvin, 2004). Once the T-DNA enters plant cytoplasm it is seen to be associated with VirE2 protein along its entire length. Vir D2 is still attached to the 5’end of T- DNA. In the cytoplasm, a plant protein VIP1 has been shown to bind to virE2. The T-complex forms a 13-15nm coiled filament and its transcytoplasmic transport is mediated by interaction of VIP1 with a dynein-like protein DLC3, the latter acting as a molecular motor for movement of the T-DNA complex along the microtubular network.

Fig 1: Transfer of T-DNA to plant cell. Transport of T-DNA to nucleus T-DNA transport to the nucleus of host plant is mediated by the bacterial effector proteins virD2 and virE2.The bacterial endonuclease virD2 remains covalently attached to the 5’ end of T-DNA, and interacts with importin α through the nuclear localization signals within virD2, thus transporting T-DNA into the nucleus. Two plant proteins are thought to play a role in modulating virD2 mediated nuclear transport of T-DNA. A protein of cyclophilin family (which are known to play

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a role in protein trafficking besides other functions) and a serine threonine phosphatase, which dephosphorylates virD2 and inhibits its nuclear import were shown to interact with virD2 and regulate T-DNA import into nucleus.

Fig 2: Transport of TDNA to nucleus. VirE2 mediated nuclear import is more complex mechanism. virE2 does not efficiently interact with the importin α, (Citovsky et al., 2004). A plant protein virE2 interacting protein 1 (VIP1), acts as an adapter for the interaction between virE2 and importin α. VIP1 import to nucleus is regulated by phosphorylation mediated by a defense related Mitogen-activated protein (MAP) kinase 3 (MPK3). MAP kinase-mediated phosphorylation of VIP1 is known to take place as a defense response in response to pathogen-associated molecular patterns (PAMPs) and phosphorylated VIP1 then enters the nucleus and induces expression of defense genes. Agrobacterium appears to hijack the VIP1 protein to gain entry into the nucleus, thus preventing its role in plant defense response. Another, Agrobacterium virulence protein virE3 partially mimics the VIP1 protein and can interact with both virE2 and importin α, facilitating the nuclear import of T-DNA. In conclusion, T-DNA transport to nucleus appears to be mediated by virD2, which may direct the T-DNA to the nuclear pore and VirE2 along with VIP1 or VirE3 may be responsible for the actual translocation of T-DNA into the nucleus. VIP1 also binds to another plant protein VIP2, which it carries to the nucleus along with the T-DNA. VIP2 is required for stable integration of T-DNA and is thought to regulate transcription of histone genes (Anand et al., 2007).

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Two other Agrobacterium virulence proteins virD5 and virF are also transported to the host cell via nuclear import machinery (Magori and Citovsky, 2011). VirF, is an F-box protein that is known to show specificity to VIP1, leading to proteosomal degradation of the latter. Hence the T-DNA complex is stripped of the proteins when it enters the nucleus. Though VirF does not recognize VirE2, it is thought to lead to removal of VirE2 as well, by destabilizing the VIP1-VirE2 complex. VirD5, which also enters the nucleus, binds to virF and protects it from proteasomal degradation by the host proteasome machinery.

Integration of T-DNA Integration of T-DNA is mainly dependent on the host DNA repair machinery. T-DNA integration was studied using Saccharomyces cerevisiae as a heterologous host for Agrobacterium (Bundock et al., 1995). In yeast, integration occurred either by non-homologous end joining (NHEJ) or by homologous recombination (HR). For HR, two host proteins, Rad52, an ssDNA-binding protein, and Rad51 (or its Arabidopsis ortholog AtRAD5) involved in homologous DNA pairing and strand exchange reaction, are required (Van Attikum and Hooykaas, 2003). Whereas for NHEJ, (a) Ku70, a

double-stranded DNA-binding protein that functions in heterodimer with Ku80, (b)DNA-dependent protein kinase (DNA-PKcs) (c) an X-ray repair cross-complementing protein 4 (XRCC4) involved in bridging DNA for ligation and (d) a DNA ligase (Lig4). DNA-PKcs and XRCC4 are anchored to Ku70 / Ku80 heterodimer, which are bound to the DNA ends. In higher plants integration mainly occurs via NHEJ method. In Arabidopsis, AtLig4 and AtKu80 were found to be needed for T-DNA integration. Rice plants showing reduced expression of Ku70, Ku80, and Lig4 showed a decrease rate of T-DNA integration. On the other hand, increasing the expression of factors required for HR pathway have resulted in increased rate of T-DNA integration. Transgenic Arabidopsis plants with higher expression of RAD54, another HR pathway protein, resulted in increased frequency of T-DNA integration by HR pathway. Other host proteins involved in T-DNA integration are those involved in chromatin structure or remodeling. Core histone protein H2A is involved in T-DNA integration (Yi et al., 2002). CAF-1, a factor involved in chromatin remodeling has proved to be limiting for T-DNA integration, since caf-1 mutants were more sensitive to stable integration than wild type plants. VIP1 also aids in T-DNA

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integration, as it forms a bridge between histone proteins and the T-complex. T-DNA is known to

become double stranded before the integration event. The second strand synthesis begins from the 3′ end of the T-strand template, most probably by random priming (Tzfira et al., 2003).

Fig 1: Model representing T-DNA integration (From: Tzfira et al., 2004).

Regulation of T-DNA expression T-DNA expression may vary among plants and is thought to depend on the ability of the plant to launch a defense response against the bacterium and the ability of the bacterium to overcome these defense responses. Regulation of the genes present in T-DNA occurs by the binding of host transcription factors to their promoters. T-DNA expression may be transient, if integration of T-DNA does not occur, or stable when T-DNA is integrated into the plant genome and is inherited during sexual reproduction. Expression of integrated T-DNA may also be limited by RNA silencing, since multiple copy insertions of T-DNA may lead to the formation of double stranded RNAs, which generate small interfering RNAs (siRNA) that target the T-DNA sequence (Sarma et al., 2015). Agrobacterium infection is shown to induce the production of salicylic acid (SA), in response to recognition of pathogen-associated molecular patterns (PAMP) by receptors located on plant cell

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membrane. SA is involved in regulating the expression of local as well as systemic acquired resistance (SAR). The expression of defense genes through the SA signaling pathway may regulate the extent of infection and T-DNA integration (Anand et al., 2008).

Summary and conclusions T-DNA entry into plant cells is mediated by the Type 4 secretion system of Agrobacterium. Besides T-DNA proteins playing a role in the formation of the T-pilus, several plant proteins are involved in attachment of the T-pilus to plant cell. Inside the cytoplasm, the T-DNA is coated by virE2 protein that also enters the plant cell via the T-pilus. A plant defense signaling protein VIP1 is recruited by the T-DNA and it binds to virE2 that coats the T-DNA. VIP2 interaction with the plant dynein proteins facilitates intro cytoplasmic movement of the T-complex. The T-DNA complex enters nucleus by interaction of importins with the nuclear localization signals present on virD2 and VIP1. An Agrobacterium F-box protein virF also enters the nucleus and brings about proteasomal degradation of VIP1 and virE2. Second strand synthesis of T-DNA takes place in the nucleus and it is then integrated into plant genomic DNA most likely by non-homologous end joining process that is a DNA repair mechanism in plant cells, involving many plant proteins. T-DNA expression in plant cells is regulated by si-RNA mediated gene silencing or by SA-mediated defense signaling. References Anand A, Uppalapathi S R, Ryu C M, Allen S N, Kang L, Tang Y, Mysore K S (2008). Salicylic acid and systemic acquired resistance play a role in attenuating crown gall disease caused by Agrobacterium tumefaciens. Plant Physiol146: 703-715.

Bundock, Paul, et al. (1995) "Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae." The EMBO journal 14: 3206.

Dumas, Fabrice, et al. (2001) "An Agrobacterium VirE2 channel for transferred-DNA transport into plant cells."Proceedings of the National Academy of Sciences 98: 485-490.

Gelvin, SB (2000) Agrobacterium and plant genes involved in T-DNA transfer and integration. Annu. Rev. Plant Physiol. Plant Mol. Biol. 51:223-256.

Hwang, H-H, and Gelvin S.B (2004)"Plant proteins that interact with VirB2, the Agrobacterium tumefaciens pilin protein, mediate plant transformation." The Plant Cell 16.11: 3148-3167.

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Lai, E-M, and Kado CI (2000)"The T-pilus of Agrobacterium tumefaciens."Trends in microbiology 8.8: 361-369.

Loyter, A, et al. "The plant VirE2 interacting protein 1. A molecular link between the Agrobacterium T-complex and the host cell chromatin?." Plant physiology 138.3 (2005): 1318-1321.

Magori, S and Citovsky V (2011)"Agrobacterium counteracts host-induced degradation of its effector F-box protein." Sci. Signal 4: ra69.

Sarma R, Pushpanathan A and Ramalingam S (2015) Epigenetic silencing in transgenic plantsFrontiers in Plant Science 6: Article693.

Tzfira T, Frankman LR, Vaidya M, and Citovsky V (2003) Site-Specific Integration of Agrobacterium tumefaciens T-DNA via Double-Stranded Intermediates. Plant Physiol.133: 1011–1023.

Tzfira T, Li J, Lacroix B, Citovsky V (2004). Agrobacterium T-DNA integration: molecules and models. Trends Genet 20: 375-383

Tzfira, T, et al. (2003)"Site-specific integration of Agrobacterium tumefaciens T-DNA via double- stranded intermediates." Plant Physiology 133.3: 1011-1023.

vanAttikum, H, et al. (2003)"The Arabidopsis AtLIG4 gene is required for the repair of DNA damage, but not for the integration of Agrobacterium T‐DNA." Nucleic acids research 31.14: 4247-4255.

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Advances in Animal and Veterinary Sciences Review Article

An Overview of Heterologous Expression Host Systems for the Production of Recombinant Proteins 1 1 Amitha Reena Gomes , Sonnahallipura Munivenkatappa Byregowda , Belamaranahally 2 3 Muniveerappa Veeregowda , Vinayagamurthy Balamurugan *

1Institute of Animal Health and Veterinary Biologicals, KVAFSU, Hebbal, Bengaluru-560024, Karnataka, India; 2Department of Microbiology, Veterinary College, KVAFSU, Hebbal, Bengaluru-560024, Karnataka, India; 3Indi- an Council of Agricultural Research-National Institute of Veterinary Epidemiology and Disease Informatics (IC- AR-NIVEDI), Post Box No. 6450, Yelahanka, Bengaluru-560064, Karnataka, India. Abstract | With the advent of recombinant DNA technology, cloning and expression of numerous mammalian genes in different systems have been explored to produce many therapeutics and vaccines for human and animals in the form of recombinant proteins. But selection of the suitable expression system depends on productivity, bioactivity, purpose and physicochemical characteristics of the protein of interest. However, large scale production of recombinant proteins is still an art in spite of increased qualitative and quantitative demand for these proteins. Researchers are constantly challenged to improve and optimise the existing expression systems, and also to develop novel approaches to face the demands of producing the complex proteins. Here, we concisely review the most frequently used conventional and alternative host systems, with their unique features, along with advantages and disadvantages and their potential appli- cations for the production of recombinant products.

Keywords | Expression host systems, Heterologous proteins, Production, Prokaryotic, Eukaryotic Editor | Kuldeep Dhama, Indian Veterinary Research Institute, Uttar Pradesh, India. Received | June 07, 2016; Accepted | June 12, 2016; Published | July 01, 2016 *Correspondence | Vinayagamurthy Balamurugan, Indian Council of Agricultural Research-National Institute of Veterinary Epidemiology and Disease Infor- matics (ICAR-NIVEDI), Post Box No. 6450, Yelahanka, Bengaluru-560064, Karnataka, India; Email: [email protected]; [email protected] Citation | Gomes AR, Byregowda SM, Veeregowda BM, Balamurugan V (2016). An overview of heterologous expression host systems for the production of recombinant proteins. Adv. Anim. Vet. Sci. 4(7): 346-356. DOI | Http://dx.doi.org/10.14737/journal.aavs/2016/4.7.346.356 ISSN (Online) | 2307-8316; ISSN (Print) | 2309-3331

Copyright © 2016 Gomes et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

xpression host systems are employed for the expres- DNA (rDNA) technology for cloning and subsequent ex- sion of recombinant proteins both for therapy and pression of particular gene of interest of virus/ microbes in Eresearch. The need for novel expression host platforms an appropriate system like bacterial / mammalian / insect increases with the number of gene-targets for various in- / yeast / plant expression systems will circumvent the dif- dustrial productions. Platforms already in use, range from ficulties associated with the production of large quantities bacteria (Baneyx, 1999), yeast (Cregg et al., 2000; Malys of vaccine or diagnostic agents (Balamurugan et al., 2006). et al., 2011) and filamentous fungi (Visser Hans, 2011) to Such a bio-engineered protein can be obtained in large cells of higher eukaryotes (Altmann et al., 1999; Kost and amounts in a pure and native form rDNA technology has Condreay, 1999). While choosing among these cells, both necessary tools to produce desired viral / bacterial proteins economic and qualitative aspects have to be considered. in a native form. The advent of rDNA technology and Industrial production depends on the use of inexpensive its application in the industry has brought about a rapid media components and high anticipated economic yield growth of biotechnology companies for the production of from the product. The quality of the product is very es- the rDNA products in human and animal healthcare. sential, especially in medicine where production of human pharmaceuticals is regulated under strict safety aspects PROKARYOTIC EXPRESSION SYSTEMS (Gellissen, 2005). Thus, suitable host system needs to be selected depending on the purpose. Use of recombinant Bacterial expression system is widely used for the expres-

NE US Academic Publishers July 2016 | Volume 4 | Issue 4 | Page 346 Advances in Animal and Veterinary Sciences sion of rDNA products. They offer several advantages viz., encounter difficulties in translating the gene, reducing the high level of recombinant protein expression, rapid cell amount of protein that is synthesized (Brown et al., 2006). multiplication and simple media requirement. However, there are some limitations such as intracellular accumu- These problems can be solved, with necessary manipula- lation of heterologous proteins, improper folding of the tions although it is time consuming. If the gene contains peptide, lack of post-transcriptional modifications, the po- introns then its complementary DNA (cDNA) prepared tential for product degradation due to trace of protease im- from the mRNA and so lacking introns might be used as purities and production of endotoxin (Balamurugan et al., an alternative. In vitro mutagenesis can be employed to 2006). Prokaryotic platforms are widely used to produce change the sequences of possible terminators and to re- recombinant proteins as these can be easily manipulated place un-favoured codons with those preferred by E. coli. genetically by well-known methods and cultivated in low An alternative with genes that are less than 1 kb in length costing medium. Among Gram-negative bacteria, E. coli is to make an artificial version. This involves synthesizing is extensively used, while Bacillus subtilis is a well-known a set of overlapping oligonucleotides that are ligated to- Gram-positive producer of recombinant proteins (Watson gether, the sequences of the oligonucleotides are designed et al., 1992). Among the several challenges protein insol- to ensure that the resulting gene contains preferred E. coli ubility and low expression levels (especially mammalian codons and terminators are absent (Brown et al., 2006). proteins) or post-translational modifications are some of the intriguing problems while using these systems (Gel- In the second category, there are three major limitations lissen, 2005). in which E. coli may prevent expression of a foreign gene. Firstly, E. coli might not process the recombinant proteins Escherichia coli correctly especially with respect to post translational mod- E. coli is a typical prokaryotic expression system and one ifications like N and O linked glycosylation, fatty acid acy- of the most attractive heterologous protein producer. The lation, phosphorylation and disulfide-bond formation that expression of proteins in this system is the easiest, quickest are required for proper folding of the secondary, tertiary and cheapest. To date reformed E. coli is the extensively and quaternary structures and for the functional character- used cellular host for foreign protein expression because istics of the protein of interest. Glycosylation is extremely of its rapid growth rate which is as short as 20-30 minutes uncommon in bacteria and recombinant proteins synthe- (Snustad and Simmons, 2010), capacity for continuous sised in E. coli are never glycosylated correctly. This can af- fermentation and relatively low cost. There are many com- fect the bioactivity, function, structure, solubility, stability, mercial and non-commercial expression vectors available half-life, protease resistance and compartmentalization of with different N and C terminal tags and many different the functional proteins ( Jung and Williams, 1997). Sec- strains are being optimized for special applications. ondly, E. coli might not correctly fold the recombinant pro- teins and is unable to synthesise disulphide bonds present There are also problems related to the use of E. coli as pro- in mammalian proteins. In such case, protein may become duction host. These problems can be grouped into two cat- insoluble and forms an inclusion body within the bacte- egories: those that are due to the sequence of the gene of rium (Leonhartsberger, 2006). Although proteins can be interest and those that are due to the limitations of E. coli as recovered from such state, converting them to correct form a host (Brown et al., 2006). In the first category again there is difficult and more often they remain inactive. Recently are three ways in which the nucleotide sequence might it has been identified that the catalytic domain of a cellu- prevent efficient expression of a foreign gene. Firstly, the lase (Cel-CD) from Bacillus subtilis can be secreted into foreign gene may contain introns which would be a major the medium from recombinant E. coli in large quantities problem since E. coli genes do not contain introns and so without its native signal peptide. It has also been proved the bacterium does not possess necessary machinery for that the N-terminal sequence of the full length Cel-CD removing introns from transcripts. Secondly, the foreign played a crucial role in transportation through both inner gene might contain sequences which act as termination and outer membranes. By subcellular location analysis, it signals in E. coli. These sequences are perfectly innocuous is identified that the secretion is a two-step process via the in the normal host cell, but in the bacterium it results in SecB-dependent pathway through the inner membrane the premature termination and a loss of gene expression. and an unknown pathway through the outer membrane Thirdly, a problem with codon bias of the gene makingE. (Gao et al., 2016). Thirdly, E. coli may degrade the recom- coli not an ideal host for translation. Although all organ- binant protein. Unlike sequence problems, these problems isms use the same genetic code, each organism has a bias can be circumvented by using mutant strains of E. coli. towards preferred codons. This bias reflects the efficiency with which the tRNA molecules are able to recognize the Accumulation of endotoxins (LPS), pyrogenic to humans different codons. If a cloned gene contains a high pro- and animals, is yet another problem in the production of portion of unfavoured codons, the host cell’s tRNAs may therapeutic proteins in E. coli, besides instability of is plas-

NE US Academic Publishers July 2016 | Volume 4 | Issue 4 | Page 347 Advances in Animal and Veterinary Sciences mid or mRNA (Terpe, 2006). Proteins expressed in E. coli organism is also used for the simultaneous coproduction also retain their amino terminal methionine, thus affecting of up to 14 recombinant proteins, multiple subsequently the stability and in turn immunogenicity of proteins (Daly interacting or forming protein complexes (Biedendieck, and Hearn, 2005). Further, expression of mammalian pro- 2016). teins in E. coli remains difficult and often results in inactive aggregates because the recombinant proteins do not fold Lactococcus lactis properly. However attempts have been made to produce Of late, Lactococcus lactis is widely used in biotechnology soluble prion proteins in E. coli (Abskharon et al., 2012). for large-scale production of heterologous proteins (Le Loir et al., 2005). During the past two decades, remarkable Bacillus subtilis progress has been made towards the development of ge- It is an alternative to the E. coli expression system. B. sub- netic engineering tools and the molecular characterization tilis, also known as hay bacillus or grass bacillus, is a Gram of L. lactis (Wegmann et al., 2007; Allain et al., 2016). positive, catalase positive bacterium, found in soil and the gastrointestinal tract of ruminants and humans. It can se- Ralstonia eutropha crete degradative enzymes or antibiotics, produce spores Ralstonia eutropha formerly known as Alcaligenes eutrophus and can become competent for genetic transformation is a Gram negative, facultative chemoautotrophic bacteri- (Maamar and Dubnau, 2005). um found in water and soil. This system overcomes some of the shortcomings of E. coli based systems, particularly It is a non-pathogenic and a GRAS (Generally regarded the formation of inclusion bodies during high-level ex- as safe) organism. The major advantage of B. subtlis is that pression (Srinivasan et al., 2002). Gavin et al. (2004) ex- it does not produce LPS, which may otherwise cause de- pressed soluble, active, organophosphohydrolase (OPH), at generative disorders in humans and animals. B. subtilis can titers greater than 10 g/L in high cell density fermentation also be transformed readily with many bacteriophages and which is approximately 100-fold greater than titers report- plasmids due to its genetic characteristics. It is capable of ed in E. coli in which it tends to form inclusion bodies. secreting functional extracellular proteins directly into the culture medium, facilitating further purification steps. It Pseudomonas has no significant bias in codon usage which is considered It is considered as a potential alternative to E. coli. Pseu- as an added advantage. Processes such as transcription, domonas fluorescens has been proven to function as a re- translation, protein folding, secretion mechanisms, genen- combinant protein producer as it can be cultivated to high tic manipulations and large scale fermentation has been cell densities often producing soluble proteins, while E. very well acquainted with this organism. The organism is coli produces insoluble protein (Squires et al., 2004). Ton- also useful for the construction of metagenomic libraries je (2011) while comparing the expression of heterologous (Luan et al., 2014). proteins P. fluorescens was found to have low stability for the expression of plasmids than P. putida which was having Like any other system this also is not without drawbacks high plasmid stability and proved to be potential recombi- and they include:- nant protein producer for industrial use with growth prop- 1. Production of extracellular proteases which recog- erties similar to E. coli during fed batch fermentations. nize and degrade heterologous proteins, but can be solved to a larger extent by construction of protease Corynebacterium deficient strains (Van Schaik and Abee, 2005; West- Non-pathogenic species of the Gram-positive Corynebac- ers et al., 2005; Servant et al., 2005). terium are used for the commercial production of various 2. Instability of plasmids which can be again overcome amino acids. The C. glutamicum is used for producing glu- by introducing plasmids using a theta mode of repli- tamate and lysine (Brinkrolf et al., 2010), components of cation ( Janniere et al., 1990; Titok et al., 2005). human food, animal feed and pharmaceutical products. 3. Reduced or non-expression of the protein of interest. Expression of functionally active human epidermal growth factor has been done in C. glutamicum (Jump et al., 2006), Bacillus megaterium thus demonstrating a potential for industrial-scale produc- Bacillus megaterium has been used for the production and tion of human proteins. secretion of recombinant proteins for many years now. Plasmids with different inducible promoter systems, with EUKARYOTIC EXPRESSION SYSTEMS different compatible origins, with small tags for protein pu- rification and with various specific signals for protein secre- In theory, prokaryotic hosts can express any gene, but in tion were combined with genetically improved host strains. practice the proteins produced do not always have the de- Along with the overproduction of individual proteins the sired biological activity or stability. Further, toxic compo-

NE US Academic Publishers July 2016 | Volume 4 | Issue 4 | Page 348 Advances in Animal and Veterinary Sciences nents from the bacteria may contaminate the final product. rification of the expressed protein (Balamurugan et al., This becomes a major issue particularly when the expressed 2006). protein is intended for therapeutic use. One must ensure that the recombinant protein must be identical to natural Presently, Pichia pastoris (Zhou et al., 2006; Ahmad et al., protein in all its properties. As eukaryotic cells share many 2014) and Schizosaccharomyces pombe (fission yeast) (Kumar molecular, genetic and biochemical features, it can be used and Singh, 2004; Benko et al., 2016) are being increasingly as an alternative to prokaryotic expression of proteins. used yeast systems for heterologous protein expression be- sides S. cerevisiae. Yeast System Yeast is an expression system with the highest commercial The enriched endomembrane system of yeasts does allow value. Among yeast, Saccharomyces cerevisiae (Baker’s yeast) some intracellularly synthesized proteins to be secreted is the most widely used. Further, it has been widely used as into the extracellular environment. As a unicellular eukar- a model organism for cell function research as its biochem- yote, yeast can produce properly folded soluble recombi- istry, genetics and cell biology is very well characterized to nant proteins with required post-translational modifica- express a number of proteins including proteins to be used tions that are essential for their functions (Daly and Hearn, for vaccines, pharmaceutical products and for diagnostics 2005). The safety of the system is guaranteed, by not having (Glick et al., 2010). It was engineered to express differ- endotoxins and oncogenes. Moreover, yeast cells are easier ent heterologous genes for almost 25 years (Hitzeman et to manipulate genetically than mammalian cells and can be al., 1981). It satisfies the economic efficiency and biosafe- grown to high cell densities. ty regulations for human applications. This system is used successfully to make hepatitis B vaccine (DiMiceli et al., But, this “lower” eukaryote differs from its mammalian 2006) and Hantavirus vaccine (Antoniukas et al., 2006). counterparts in the way it forms both N and O linked oli- It has been used to optimize the functional yields of po- gosaccharide structures on target proteins ( Jung and Wil- tential antigens for the development of subunit vaccines liams, 1997). Particularly, S. cerevisiae is unable to glyco- against a wide range of diseases caused by bacteria and vi- sylate animal proteins correctly and it may often add many ruses. Saccharomyces cerevisiae is used in the manufacture sugars leading to hyper-glycosylation of proteins. Recent- of 11 approved vaccines against hepatitis B virus and one ly, advances in the glycoengineering of yeast and the ex- against human papillomavirus; in both cases, the recombi- pression of therapeutic glycoproteins with humanized N nant protein forms highly immunogenic virus-like parti- glycosylation structures have shown a significant promise cles (Bill, 2015). (Wildt and Gerngross, 2005). Besides codon biasness, yeast system is an inefficient one in secreting the proteins into Among the eukaryotic systems, yeast is unique in that it growth medium leading to intracellular retention making combines the advantages of both prokaryotic [high expres- them lot more difficult to purify. sion levels (10-100 fold higher), faster growth, easy main- tenance, easy scale-up, inexpensive growth media] and Pichia pastoris makes a good alternative with glycosylation eukaryotic (capacity to carry out most of the post-transla- abilities similar to those of animal cells. Even though, the tional modifications like protein processing, protein fold- sugar structures it synthesizes are not same as the animal ing etc.,) expression systems. Yeast such as Saccharomyces versions but the differences are relatively trivial and does cerevisiae, Hansenulla polymorpha and P. pastoris are among not have a significant effect on the activity of a recombi- the simplest eukaryotic organisms, which grow relatively nant protein. quickly and are highly adaptable to large-scale production, Technical advantages in this system include site-specific Meanwhile, several “non-conventional” yeasts (Gellissen et integration, increase in copy number, leader sequence for al., 2005) are well established as expression systems, which the secretion of heterologous protein and post-translation- include Arxula adeninivorans (Terentiev et al., 2004), al modifications (Balamurugan et al., 2006). Hansenula polymorpha (Kulkarni et al., 2006), Kluyvero- myces lactis (Donnini et al., 2004) and Yarrowia lipolytica In recent past, the methylotrophic yeasts, such as (Madzak et al., 2004). Hansenulla polymorpha, Pichia pastoris and Candid- ia biodini have been developed, among which P. p a s - Fungus System toris has emerged as a powerful and inexpensive het- Filamentous fungi, especially Aspergillus (Aspergillus niger erologous system for the production of high levels of and Aspergillus oryzae) and Trichoderma have been de- functionally active recombinant proteins (Balamurugan veloped into expression platforms for screening and pro- et al., 2007), in addition to existing Saccharomyces. Intact duction of diverse industrial enzymes. Trichoderma reesei protein production and secretion into the medium makes has tremendous capability to secrete over 100 g/L of pro- yeast could be an efficient system for production and pu- teins and therefore makes an excellent host system for pro-

NE US Academic Publishers July 2016 | Volume 4 | Issue 4 | Page 349 Advances in Animal and Veterinary Sciences duction of high levels of therapeutic proteins at low cost er. The recombinant viruses are selected by their inability (Landowski et al., 2016). Fungal-based systems have several to induce inclusion body formation 72 hrs after infection advantages due mainly to their high-level secretion of en- of the cell lines or infecting insects for the production of zymes and their decomposer lifestyle. Further, in the large- recombinant protein. The production level in this system scale production of recombinant proteins of eukaryotic is very high and if the recombinant virus is prepared for origin, the filamentous fungi become the system of choice infecting insects then the production of protein is very due to critical processes shared in gene expression with cheap. However, there is one drawback with this system other eukaryotic organisms. But, the complexity and rela- that glycosylation of protein in insect cells is different from tive dearth of understanding the physiology of filamentous mammalian cells which leads to improper maintenance of fungi, compared to bacteria, have hindered rapid develop- epitopes in the target protein (Balamurugan et al., 2006). ment of these organisms as highly efficient factories for the production of heterologous proteins (Su et al., 2012). More Baculoviruses are insect pathogens that regulate insect recently Myceliophthora thermophila, C1 (Visser Hans, populations in nature and are being successfully used to 2011) has been developed into an expression platform for control insect pests. Typical property of very late gene ex- screening and production of diverse industrial enzymes. pression makes them highly suitable as vectors for foreign C1 shows a less viscous morphology in submerged culture, gene expression. Two baculoviruses are broadly applied in enabling the use of complex growth and production media. biotechnology as vectors to produce recombinant proteins T. reesei strains suitable for production of therapeutic pro- in insect cells: Autographa californica multiple nucleopoly- teins by reducing the secreted protease activity have also hedrovirus (AcMNPV) and to a lesser extent Bombyx mori been developed recently (Landowski et al., 2016). (Bm) NPV. An additional advantage is that they have a limited insect host range and hence safe for vertebrates. Insect System Insect cells can grow in serum-free media and the cultures Insect cell culture systems are widely used for the produc- can easily be scaled up (Smagghe et al., 2009; van Oers tion of recombinant proteins, vaccines and viral pesticides and Lynn, 2010). The lepidopteran insect cells are also free as well as in the basic research in biology. A large number of human pathogens. The proteins produced in the bacu- of cell lines from diptera, hemiptera and lepidopteran in- lovirus-expression system are used for functional studies, sects have been established. High levels of heterologous vaccine preparations or diagnostics. gene expression are often achieved compared to other eu- karyotic expression systems, particularly for intracellular The first vaccine produced in the baculovirus expression proteins (Balamurugan et al., 2006). In many cases, the system, which was commercialized and approved, is direct- recombinant proteins are soluble and easily recovered from ed against classical swine fever or hog cholera (Intervet) infected cells late in the infection when host proteins syn- and is accompanied by a serological test (Bouma et al., thesis is diminished. Insect cell based systems especially 1999; van Rijn et al., 1999) thus allowing the differenti- baculovirus based systems revolutionized the recombinant ation between infected and vaccinated animals. The sys- protein production. Baculovirsuses have a restricted host tem is valuable for the production of proteins for structural range limited to specific invertebrate species. Being nonin- studies and G protein-coupled receptors. It is also used for fectious to vertebrates, these viruses are safer to work with production of the human papilloma virus vaccine, Cervar- than most mammalian viruses. Most of the susceptible in- ix, the first FDA approved insect cell produced product sect cell lines are not transformed with pathogenic or infec- and FluBlok, a vaccine based on the influenza virus he- tious viruses and can be cared for under minimal contain- magglutinin protein. MultiBac, an advanced baculovi- ment conditions. Helper cell lines or helper viruses are not rus system, has been widely adopted in the last decade to needed since the baculovirus genome contains all the ge- produce multiprotein complexes with many subunits that netic information needed for propagation in a variety of were hitherto inaccessible, for academic and industrial re- cell lines or larvae from different insects. Baculoviruses search and development (Sari et al., 2016). Baculovirus- are usually propagated in insect cell lines derived from es, modified to contain mammalian promoters (BacMam the fall armyworm, Spodoptera frugiperda or from the viruses), have proven to be efficient gene delivery vectors cabbage looper, Trichoplusia ni. Commonly used and for mammalian cells and provide an alternative transient commercially available insect cell lines are Sf-9, Sf-21 and mammalian cell based protein expression approach to that high five tTrichoplusia ni.). Prolific cell lines are available of plasmid DNA based transfection methodologies (Kost which grow well in suspension cultures, permitting the and Kemp, 2016). production of recombinant proteins in large-scale bioreac- tors. The recombinant proteins can be produced in insect One of the most appealing features of baculovirus–insect cell lines as well as in insects. This recombinant virus can expression systems has been the eukaryotic protein pro- be prepared by cloning any DNA insert coding protein of cessing capabilities of the host. Accordingly, these systems desire under polyhedron growth promoter or Pro promot- are widely considered to be excellent tools for recombinant

NE US Academic Publishers July 2016 | Volume 4 | Issue 4 | Page 350 Advances in Animal and Veterinary Sciences glycoprotein production. Nearly a thousand of high-val- and BHK cell systems are the ideal choice for production ue foreign proteins have been successfully produced in the of therapeutic proteins as these are capable of glycosylating system and the insect baculovirus may be applied in pro- the protein at the correct sites. However, cost of production duction of vaccines (Kang, 1997), gene therapy (Ghosh et of the products using these cell systems is high because of al., 2002) and recombinant baculovirus insecticides (As- the slow growth and expensive nutrient requirement. The senga et al., 2006). choice of an expression system invariably influences the character, quantity and cost of a final product. Like any other system, this system is also not free from limitations. As the host cell infected with nuclear poly- Further, recent advances have been made in producing hedrosis virus will eventually die, the heterologous gene therapeutic proteins by using transgenic animates. Eukar- cannot be expressed continuously. Every round of synthe- yotic individuals systems are a newly emerging expression sis of the protein of interest requires the infection of new system, which includes both individual animal and indi- insect cells. Therefore, this system is inferior to prokaryotic vidual plant expression systems. and yeast systems in terms of its capacity for continuous fermentation. Moreover, insect cells and mammalian cells Transgenic Plants differ in their glycosylation patterns, such as in the lengths The plants can be considered as a solar- powered biore- of oligosaccharides and in mannose content (Kost and actor and proved to be advantageous over the alternative Condreay, 1999; Marheineke et al., 1998), so the bioac- fermentation systems of biomass production using mi- tivity and immunogenicity of insect expression products crobial or animal cells. The requirements for plant system are somewhat different from those of the natural product. are rather simple and inexpensive. Plants, being eukaryot- However, people have successfully addressed this limita- ic, are also capable of the post-translational processing of tion by genetically transforming established lepidopteran proteins of eukaryotic origin, which may be essential to insect cell lines with constitutively expressible mammalian their proper functioning. The complex, multi-meric pro- genes. This approach has yielded transgenic insect cell lines teins can be readily assembled in plant cells and individual with normal growth properties that can support baculovi- plant expressing genes encoding different components of rus infection, having new N glycan processing enzyme ac- multi-meric complexes can be readily obtained by sexual tivities, producing humanized recombinant glycoproteins crossing of plants harbouring a single transgene. The use of (Donald, 2003). genetically engineered plants to produce valuable proteins is increasing slowly. The system has potential advantages of Mammalian Systems economy and scalability. However, variations product yield, There appears to be a progressive increase in the application contamination with agrochemical and fertilizers, impact of of mammalian cells for protein production. Expression sys- pest and disease and variable cultivation conditions should tems utilizing mammalian cells for recombinant proteins also be considered. Plant cell culture system combines the are able to introduce proper protein folding, post-transla- advantages of whole plant system as well as animal cell tional modifications and product assembly, which are im- culture, Although no recombinant products have yet been portant for complete biological activity (Khan, 2013). They produced commercially using plant cell culture several also promote signal synthesis, process and can secrete and companies are investigating the commercial feasibility of glycosylate proteins, particularly eukaryotic proteins. such a production system (Balamurugan et al., 2006).

A number of mammalian cell lines have been utilized for Plant cells share some architectural and functional simi- protein expression with the most common being HEK 293 larities with animal cells. In particular, they constitute an (Human embryonic kidney) and CHO (Chinese ham- optimal system to express heterologous proteins that re- ster ovary). These cell lines can be transfected using pol- quire complicated post-translational modifications, such as yethyleneimine (PEI) or calcium phosphate. Apart from some glycoproteins, bioactive peptides, and drugs. Unlike this, baby hamster kidney (BHK) cells, Vero cells, mouse most other expression systems, individual plant expression L-cells and myeloma cell lines like J558L and Sp2/0, etc., systems have greater and distinctive flexibility and utility. are also employed as hosts for the establishment of stable Firstly, the heterologous proteins expressed can be localized transfectants (Geisse et al., 1996; Castro et al., 2014). Even to different organs of the plant by controlling the tissue-spe- though, the high cost, complicated technology and poten- cific regulatory sequences involved in gene expression. Sec- tial contamination with animal viruses have been the lim- ondly, proteins can be expressed at specific growth stages itations for its use in large-scale industrial production, this by manipulating the inducible components and develop- system is often utilized to express many heterologous pro- ment-specific regulatory sequences. Thirdly, plants can be teins including viral structural protein and bioactive pep- grown in the field, providing a very inexpensive source of tide for specific functional analysis because of its advantag- material compared to any organism that needs to be grown es (Nagpal et al., 2004). Mammalian systems such as CHO in fermentors. Thus scale-up of plant-based expression

NE US Academic Publishers July 2016 | Volume 4 | Issue 4 | Page 351 Advances in Animal and Veterinary Sciences Table 1: Merits and demerits of different host systems for expression of recombinant proteins Host system Merits Demerits Escherichia coli Easy Does not possess necessary machinery for removing in- Quick trons from transcripts Economical Foreign gene might contain sequences that act as termina- Rapid growth rate tion signals resulting in premature termination and loss of Capacity for continuous fermentation gene expression Codon bias Lack of post translational modifications Glycosylation is extremely uncommon in bacteria Production of proteins in the insoluble form or in the form of inclusion bodies Degradation of proteins Accumulation of endotoxins Bacillus subtilis Does not produce LPS/endotoxins Production of extracellular proteases which can recognize Can be transformed readily with many bacteri- and degrade heterologous proteins ophages and plasmids Instability of plasmids Capable of secreting functional extracellular Reduced or non expression of the protein of interest proteins directly into the culture medium Yeast system Rapid growth in low cost medium Hyperglycosylation of proteins Appropriate post-translational modifications Codon bias Safety of the system is guaranteed Inefficient in secreting the proteins into growth medium No endotoxins production leading to intracellular retention Filamentous High-level of expression Complex fungus Lack of knowledge on physiology Baculovirus / High level of expression Continuous expression not possible Insect system Appropriate posttranslational modifications More demanding culture conditions Safe for vertebrates Excellent tool for recombinant glycoprotein production Mammalian cells Proper protein folding High cost / system Appropriate post-translational modifications Complicated technology and product assembly Potential contamination with animal viruses Proper glycosylation Transgenic Easy scaling up at low cost Expression levels are target dependent plants Proteins can be localized to different organs at Functional assays yet to be developed different growth stages High yield Transgenic Proper protein folding Relatively longer production period animals Appropriate post-translational modifications Low yield and product assembly Higher costs Proper glycosylation is much easier than in other systems: more plants can be of choice to express valuable recombinant proteins because grown easily, reliably increasing total yield, compared to milk is easily collected in large volumes and is the best avail- culture and fermentor reactor-based systems that are often able bioreactor. Foreign proteins are commonly reported very difficult to scale up (Chen and Davis, 2016). Finally, to be produced in transgenic milk at rates of several grams expression of proteins in plant seeds results in a unit of per litter. Apart from milk, egg white, blood, urine, seminal production (seed), in which proteins are extremely stable, plasma and silkworm cocoon are the alternative systems readily stored, and easily extracted and purified (Yin et al., available for the production of useful pharmaceutical pro- 2007; Yemets et al., 2014). teins. The various mammals used as bioreactors are rabbits, pigs, sheep, goats and cows. Each of these species offers ad- Transgenic Animals vantages and drawbacks. Rabbits are sufficient to produce Transgenic animal bioreactors can produce therapeu- several kilograms of proteins per year. The rabbit is par- tic proteins with high value for pharmaceutical use. The ticularly flexible, allowing rapid generation and scaling-up. mammary gland has generally been considered the organ For very high protein production, larger animals are needed

NE US Academic Publishers July 2016 | Volume 4 | Issue 4 | Page 352 Advances in Animal and Veterinary Sciences

Figure 1: Different host systems available for the production of recombinant Proteins

(Wang et al., 2013). However, the individual animal ex- quantity of the protein, cost, availability, convenience and pression system requires a relatively longer production pe- purposes of the expressed products. Further, for functional riod with low yield and higher costs than above-mentioned analysis and preparation of vaccines eukaryotic system can expression systems. So this system can express foreign pro- be considered and for production of artificial antigens for teins mainly for medical purposes (Yin et al., 2007). diagnostic purposes or for studying the structural analy- sis of particular proteins that require no post-translational In nutshell, the different host systems available for expres- modification, prokaryotic system can be selected. Products sion of recombinant proteins and their merits and demerits developed in the field of veterinary medicine will be most are summarized (Figure 1 and Table 1). Recombinant pro- valuable for further development of rDNA products teins are being produced using either of the heterologous in the coming decades. In view of high market po- systems mentioned above and have a potential value in the tential for recombinant therapeutics as the case with development of diagnostic test as well as vaccines for the human therapeutics, indigenous technology should prophylaxis of various infectious diseases of human and also be developed for the veterinary field to develop a veterinary importance (Balamurugan et al., 2006). A hall- prophylactics and diagnostics. This can be achieved by mark in the prevention and control of several diseases of the strengthening the linkages among various institutes having animals and human used by viruses, bacteria and parasites expertise in different disciplines related to rDNA technol- is the development of suitable vaccines against infectious ogy and increased interaction with the industry. diseases. So far, various attempts have been made in the recent past to produce antigens in heterologous systems ACKNOWLEDGEMENTS for use as diagnostics as well as prophylactics. Biologically active peptides and proteins have many potential applica- All the authors who contributed to the subject irrespective tions including being used as vaccines, immuno-modula- of their citation being figured or not are acknowledged. tors, growth factors, hormones and enzymes.

CONCLUSIONS CONFLICT OF INTEREST

In general, rDNA technology has indeed made tremen- No conflicts of interests are declared by authors for the dous breakthrough in the discovery of various recombi- contents in this manuscript. nants antigens or proteins. The new generation proteins prepared from the viral/microbial proteins; their frag- AUTHORS’ CONTRIBUTION ments or the nucleic acid sequences have been attractive because of their stability, non-infectious nature, homoge- A. R. Gomes drafted the manuscript, S. M. Byregowda and neity as well as their cost-effectiveness. One should care- B. M. Veeregowda gave the technical guidance. V. Bala- fully choose the system for a specific expression procedure murugan provided technical guidance, inputs and edited considering bio-characteristics of the protein, quality and the manuscript.

NE US Academic Publishers July 2016 | Volume 4 | Issue 4 | Page 353 Advances in Animal and Veterinary Sciences REFERENCES • Brown TA (2006). Gene cloning and DNA Analysis, An Introduction. Blackwell Publishing. Pp. 277-301 • Castro PV, de Freitas MC, Bomfim Ade S, de Sousa Russo • Abskharon RNN, Ramboarina S, Hassan HE, Gad W, Apostol EM (2014). Patents in therapeutic recombinant protein MI, Giachin G, Legname G, Steyaert J, Messens J, Soror production using mammalian cells. Recent Pat. Biotechnol. H, Wohlkonig A (2012). A novel expression system for 8(2): 165-71. http://dx.doi.org/10.2174/187220830966614 production of soluble prion proteins in E. coli. Microb. Cell 0904120404 Fact. 11: 6. http://dx.doi.org/10.1186/1475-2859-11-6 • Chen Q, Davis KR (2016). The potential of plants as a • Ahmad M, Hirz M, Pichler H, Schwab H (2014). Protein system for the development and production of human expression in Pichia pastoris: recent achievements and biologics. F1000Res. 19: 5. http://dx.doi.org/10.12688/ perspectives for heterologous protein production. Appl. f1000research.8010.1 Microbiol. Biotechnol. 98(12): 5301-17. http://dx.doi. • Cregg JM, Cereghino JL, Shi J, Higgins DR (2000). Recombinant org/10.1007/s00253-014-5732-5 protein expression in Pichia pastoris. Mol. Biotechnol. 16 (1): • Allain T, Mansour NM, Bahr MM, Martin R, Florent I, Langella 23–52. http://dx.doi.org/10.1385/MB:16:1:23 P, Bermúdez-Humarán LG (2016). A new lactobacilli in • Daly R, Hearn MT (2005). Expression of heterologous proteins vivo expression system for the production and delivery of in Pichia pastoris: a useful experimental tool in protein heterologous proteins at mucosal surfaces. FEMS Microbiol. engineering and production, J Mol Recognit. 18: 119-38. Lett. 363(13). http://dx.doi.org/10.1093/femsle/fnw117 http://dx.doi.org/10.1002/jmr.687 • Altmann F, Staudacher E, Wilson IB, März L (1999). • DiMiceli L, Pool V, Kelso JM, Shadomy SV, Iskander J Insect cells as hosts for the expression of recombinant (2006). Vaccination of yeast sensitive individuals: review glycoproteins. Glycoconjugate J. 16 (2): 109–23. http:// of safety data in the US vaccine adverse event reporting dx.doi.org/10.1023/A:1026488408951 system (VAERS). Vaccine. 24: 703–707. http://dx.doi. • Antoniukas L, Grammel H, Reichl U (2006). Production of org/10.1016/j.vaccine.2005.07.069 Hantavirus Puumala nucleocapsid protein in Saccharomyces • Donnini C, Farina F, Neglia B, Compagno MC, Uccelletti D, cerevisiae for vaccine and diagnostics. J. Biotechnol. 124: Goffrini P, Palleschi C (2004). Improved production of 347–362. http://dx.doi.org/10.1016/j.jbiotec.2005.12.028 heterologous proteins by a glucose repression-defective • Assenga SP, You M, Shy CH, Yamagishi J, Sakaguchi T, mutant of Kluyveromyces lactis. Appl. Environ. Microbiol. 70: Zhou J, Kibe MK, Xuan X, Fujisaki K (2006). The use of a 2632–2638. http://dx.doi.org/10.1128/AEM.70.5.2632- recombinant baculovirus expressing a chitinase from the hard 2638.2004 tick Haemaphysalis longicornis and its potential application as • Donald L Jarvis (2003). Developing baculovirus-insect cell a bioacaricide for tick control. Parasitol. Res. 98: 111–118. expression systems for humanized recombinant glycoprotein http://dx.doi.org/10.1007/s00436-005-0007-9 production. Virol. 310: 1–7. http://dx.doi.org/10.1016/ • Balamurugan V, Sen A, Saravanan P, Singh RK (2006). s0042-6822(03)00120-x Biotechnology in the Production of Vaccine or Antigen for • Gao D, Luan Y, Liang Q, Qi Q (2016). Exploring the N-terminal animal health. J. Anim. Vet. Adv. 5 (6): 487-495. role of a heterologous protein in secreting out of Escherichia • Balamurugan V, Reddy GR, Suryanarayana VVS (2007). Pichia coli. Biotechnol. Bioeng. 9999: 1-7. pastoris: a notable heterologous expression system for the • Gavin CB, Henderson GE, Sriram Srinivasan, Tillman U, production of foreign proteins-Vaccine. Indian J. Biotech. 6: Gerngross (2004) High level recombinant protein expression 175-186. in Ralstonia eutropha using T7 RNA polymerase based • Baneyx F (1999). Recombinant protein expression in Escherichia amplification. Protein Expr. Purif. 38: 264-71. http://dx.doi. coli. Curr. Opin. Biotechnol. 10(5): 411–21. http://dx.doi. org/10.1016/j.pep.2004.09.001 org/10.1016/S0958-1669(99)00003-8 • Geisse S, Gram H, Kleuser B, Kocher HP (1996). Eukaryotic • Benko Z, Elder RT, Li G, Liang D, Zhao RY (2016). HIV-1 expression systems: a comparison. Protein Expr. Purif. 8: Protease in the Fission Yeast Schizosaccharomyces pombe. 271–282. PLoS One. 11(3). http://dx.doi.org/10.1371/journal. • Gellissen G (ed.) (2005). Production of Recombinant Proteins: pone.0151286 Novel Microbial and Eukarytoic Expression Systems: • Biedendieck R (2016). A Bacillus megaterium system for Wiley-VCH. the production of recombinant proteins and protein • Ghosh S, Parvez MK, Banerjee K, Sarin SK, Hasnain SE, complexes. Adv. Exp. Med. Biol. 896: 97-113. http://dx.doi. (2002). Baculovirus as mammalian cell expression vector org/10.1007/978-3-319-27216-0_7 for gene therapy: an emerging strategy. Mol. Ther. 6: 5–11. • Bill RM (2015). Recombinant protein subunit vaccine synthesis http://dx.doi.org/10.1006/mthe.2000.0643 in microbes: A role for yeast? J. Pharm. Pharmacol. 67(3): • Glick BR, Pasternak JJ, Patten CL (2010). Molecular 319-28. http://dx.doi.org/10.1111/jphp.12353 Biotechnology, Washington, American Society for • Bouma A, de Smit AJ, de Kluijver EP, Terpstra C, Moormann Microbiology. RJ (1999). Efficacy and stability of a subunit vaccine based • Hitzeman RA, Hagie FE, Levine HL, Goeddel DV, Ammerer on glycoprotein E2 of classical swine fever virus. Vet. G, Hall BD (1981). Expression of a human gene for Microbiol. 66: 101–114. http://dx.doi.org/10.1016/s0378- interferon in yeast. Nat. 293: 717–722. http://dx.doi. 1135(99)00003-6 org/10.1038/293717a0 • Brinkrolf K, Schröder J, Pühler A, Tauch A (2010). The • Itakura K, Hirose T, Crea R, Riggs AD, Heyneker HL, Bolivar transcriptional regulatory repertoire of Corynebacterium F, Boyer HW (1977). Expression in Escherichia coli of a glutamicum: reconstruction of the network controlling chemically synthesized gene for the hormone somatostatin. pathways involved in lysine and glutamate production. Sci. 198: 1056–1063. http://dx.doi.org/10.1126/ J. Biotechnol. 149: 173-82. http://dx.doi.org/10.1016/j. science.412251 jbiotec.2009.12.004

NE US Academic Publishers July 2016 | Volume 4 | Issue 4 | Page 354 Advances in Animal and Veterinary Sciences • Jannière L, Bruand C, Ehrlich SD (1990). Structurally stable Trichoplusia ni (Tn) insect cells used for baculovirus Bacillus subtilis cloning vectors. Gene. 87: 53-61. http:// infection. FEBS Lett. 441: 49-52. http://dx.doi.org/10.1016/ dx.doi.org/10.1016/0378-1119(90)90495-D S0014-5793(98)01523-3 • Jump up Date M, Yokoyama K, Umezawa Y, Matsui H, Kikuchi • Nagpal ML, Chen Y, Lin T (2004). Effects of overexpression Y (2006). Secretion of human epidermal growth factor by of CXCL10 (cytokine-responsive gene-2) on MA-10 Corynebacterium glutamicum. Lett. Appl. Microbiol. 42: 66- mouse Leydig tumor cell steroidogenesis and proliferation. 70. J. Endocrinol. 183: 585–594. http://dx.doi.org/10.1677/ • Jung E, Williams KL (1997). The production of joe.1.05795 recombinant glycoproteins with special reference to • Sari D, Gupta K, Raj DB, Aubert A, Drncová P, Garzoni F, simple eukaryotes including Dictyostelium discoideum. Fitzgerald D, Berger I (2016). The MultiBac Baculovirus/ Biotechnol. Appl. Biochem. 25: 3–8. http://dx.doi. Insect Cell Expression Vector System for Producing org/10.1111/j.1470-8744.1997.tb00407.x Complex Protein Biologics. Adv. Exp. Med. Biol. 896: 199- • Kang CY (1997). Expression of human immunodeficiency virus 215. http://dx.doi.org/10.1007/978-3-319-27216-0_13 genes using baculovirus expression system. Mol. Biotechnol. • Servant P, Le Coq D, Aymerich S (2005). CcpN (YqzB), a 8: 173–187. http://dx.doi.org/10.1007/BF02752262 novel regulator for CcpA-independent catabolite repression • Khan KH (2013). Gene Expression in Mammalian Cells and its of Bacillus subtilis gluconeogenic genes. Mol. Microbiol. Applications. Adv. Pharm. Bull. 3: 257–263. 55: 1435–1451. http://dx.doi.org/10.1111/j.1365- • Kost TA, Condreay JP (1999). Recombinant baculoviruses as 2958.2005.04473.x expression vectors for insect and mammalian cells. Curr. • Smagghe G, Goodman CL, Stanley D (2009). Insect cell culture Opin. Biotechnol. 10: 428-33. http://dx.doi.org/10.1016/ and applications to research and pest management. In vitro S0958-1669(99)00005-1 cellular & developmental biology. Anim. 45: 93–105. http:// • Kulkarni PS, Raut SK, Patki PS, Phadke MA, Jadhav SS, Kapre dx.doi.org/10.1007/s11626-009-9181-x SV, Dhorje SP, Godse SR (2006). Immunogenicity of a • Snustad DP, Simmons MJ (2010). Principles of Genetics, Wiley. new, low-cost recombinant hepatitis B vaccine derived from • Srinivasan S, Barnard GC, Gerngross TU (2002). A novel high- Hansenula polymorpha in adults. Vaccine. 24: 3457–3460. cell-density protein expression system based on Ralstonia http://dx.doi.org/10.1016/j.vaccine.2006.02.008 eutropha. Appl. Environ. Microbiol. 68: 5925–5932. http:// • Kumar R, Singh, J, (2004). Expression and secretion of a dx.doi.org/10.1128/AEM.68.12.5925-5932.2002 prokaryotic protein streptokinase without glycosylation and • Su X, Schmitz G, Zhang M, Mackie RI, Cann IK (2012). degradation in Schizosaccharomyces pombe. Yeast. 21: 1343– Heterologous gene expression in filamentous fungi. Adv. 1358. http://dx.doi.org/10.1002/yea.1184 Appl. Microbiol. 81: 1-61. http://dx.doi.org/10.1016/B978- • Landowski CP, Mustalahti E, Wahl R, Croute L, Sivasiddarthan 0-12-394382-8.00001-0 D, Westerholm-Parvinen A, Sommer B, Ostermeier C, • Squires CH, Retallack DM, Chew LC, Ramseier TM, Schneider Helk B, Saarinen J, Saloheimo M (2016). Enabling low JC, Talbot HW (2004). Heterologous Protein Production in cost biopharmaceuticals: high level interferon alpha-2b P. fluorescens. Bio Process Int. 2: 54-59. production in Trichoderma reesei. Microb. Cell Fact. 15(1): • Terentiev Y, Pico AH, Boe RE, Wartmann T, Klabunde J, Breuer 104. http://dx.doi.org/10.1186/s12934-016-0508-5 U, Babel W, Suckow M, Gellissen G, Kunze G (2004). A • Le Loir Y, Azevedo V, Oliveira SC, Freitas DA, Miyoshi A wide-range integrative yeast expression vector system based (2005). Protein secretion in Lactococcus lactis: an efficient on Arxula adeninivorans-derived elements. J. Ind. Microbiol. way to increase the overall heterologous protein production. Biotechnol. 31: 223–228. http://dx.doi.org/10.1007/ Microb. Cell Fact. 4: 2. http://dx.doi.org/10.1186/1475- s10295-004-0142-9 2859-4-2 • Terpe, K. (2006). Overview of bacterial expression systems • Leonhartsberger S (2006). E.coli Expression System Efficiently for heterologous protein production: from molecular Secretes Recombinant Proteins into Culture Broth. and biochemical fundamentals to commercial systems. BioProcess International. Appl. Microbiol. Biotechnol. 72: 211–222. http://dx.doi. • Luan C, Zhang HW, Song de G, Xie YG, Feng J, Wang YZ org/10.1007/s00253-006-0465-8 (2014). Expressing antimicrobial peptide cathelicidin-BF in • Titok MA, Chapuis J, Selezneva YV, Lagodich AV, Prokulevich Bacillus subtilis using SUMO technology. Appl. Microbiol. VA, Ehrlich SD, Jannière L (2003). Bacillus subtilis soil Biotechnol. 98: 3651-58. http://dx.doi.org/10.1007/s00253- isolates: plasmid replicon analysis and construction of a new 013-5246-6 theta-replicating vector. Plasmid. 49: 53-62. http://dx.doi. • Maamar H, Dubnau D (2005). Bistability in the Bacillus org/10.1016/S0147-619X(02)00109-9 subtilis K-state (competence) system requires a positive • Tonje Husby Haukaas (2011). Development of Expression feedback loop. Mol. Microbiol. 56: 615–624. http://dx.doi. Systems and Cultivation Conditions for Production of org/10.1111/j.1365-2958.2005.04592.x Heterologous Proteins in Pseudomonas. Thesis Norwegian • Madzak C, Gaillardin C, Beckerich JM (2004). Heterologous University of Science and Technology. protein expression and secretion in the non-conventional • Van Oers MM, Lynn DE (2010). Insect Cell Culture in yeast Yarrowia lipolytica: a review. J. Biotechnol. 8: 63–81. Encycopledia of Life Science. Elsevier. http://dx.doi.org/10.1016/j.jbiotec.2003.10.027 • Van Schaik W, Abee T (2005). The role of sigma (B) in the • Malys N, Wishart JA, Oliver SG, McCarthy JE (2011). Protein stress response of Gram-positive bacteria—targets for food production in Saccharomyces cerevisiae for systems biology preservation and safety. Curr. Opin. Biotechnol. 16: 218– studies. Methods Enzymol. 500: 197–212. http://dx.doi. 224. http://dx.doi.org/10.1016/j.copbio.2005.01.008 org/10.1016/B978-0-12-385118-5.00011-6 • Van Rijn PA, van Gennip HG, Moormann RJ (1999). An • Marheineke K, Grünewald S, Christie W, Reiländer H (1998). experimental marker vaccine and accompanying serological Lipid composition of Spodoptera frugiperda (Sf9) and diagnostic test both based on envelope glycoprotein E2 of

NE US Academic Publishers July 2016 | Volume 4 | Issue 4 | Page 355 Advances in Animal and Veterinary Sciences classical swine fever virus (CSFV). Vaccine. 17: 433–440. WJ (2005). Genes involved in SkfA killing factor production http://dx.doi.org/10.1016/S0264-410X(98)00215-1 protect a Bacillus subtilis lipase against proteolysis. Appl. • Visser H, Joosten V, Punt PJ, Gusakov AV, Olson PT, Joosten R, Environ. Microbiol. 71: 1899–1908. http://dx.doi. Bartels J, Visser J, Sinitsyn AP, Emalfarb MA, Verdoes JC, org/10.1128/AEM.71.4.1899-1908.2005 Wery J (2011). Development of a mature fungal technology • Wildt S, Gerngross TU (2005). The humanization of and production platform for industrial enzymes based on Nglycosylation pathways in yeast. Nat. Rev. Microbiol. 3: a Myceliophthora thermophila isolate, previously known 119–128. http://dx.doi.org/10.1038/nrmicro1087 as Chrysosporium lucknowense C1. Indust. Biotechnol. 7: • Yemets AI, Tanasienko IV, Krasylenko YA, Blume YB (2014). 214-223. http://dx.doi.org/10.1089/ind.2011.7.214 Plant-based biopharming of recombinant human lactoferrin. • Wang Y, Zhao S, Bai L, Fan J, Liu E (2013). Expression systems Cell Biol. Int. 38(9): 989-1002. http://dx.doi.org/10.1002/ and species used for transgenic animal bioreactors. Biomed. cbin.10304 Res. Int. 1-9. http://dx.doi.org/10.1155/2013/580463 • Yin J, Guangxing Li, Xiaofeng Ren, Georg Herrler (2007). Select • Watson JD, Gilman M, Witkowski J, Zoller M (1992). what you need: A comparative evaluation of the advantages Recombinant DNA, W.H.Freeman & Co Ltd. and limitations of frequently used expression systems for • Wegmann U, O’Connell-Motherway M, Zomer A, Buist G, foreign genes. J. Biotechnol. 127: 335–347. http://dx.doi. Shearman C (2007). Complete genome sequence of the org/10.1016/j.jbiotec.2006.07.012 prototype lactic acid bacterium Lactococcus lactis subsp. • Zhou JM, Tang YX, Fang DY, Zhou JJ, Liang Y, Guo HY, Jiang Cremoris MG1363. J. Bacteriol. 189: 3256–3270. http:// LF (2006). Secreted expression and purification of dengue dx.doi.org/10.1128/JB.01768-06 2 virus full-length nonstructural glycoprotein NS1 in Pichia • Westers H, Braun PG, Westers L, Antelmann H, Hecker M, pastoris. Virus Genes. 33: 27–32. http://dx.doi.org/10.1007/ Jongbloed JD,Yoshikawa H, Tanaka T, Van Dijl J.M, Quax s11262-005-0036-6

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Expression of heterologous genes in plant systems: New possibilities

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The user has requested enhancement of the downloaded file. ISSN 10227954, Russian Journal of Genetics, 2012, Vol. 48, No. 11, pp. 1067–1079. © Pleiades Publishing, Inc., 2012. Original Russian Text © A.O. Vyacheslavova, I.N. Berdichevets, A.A. Tyurin, Kh.R. Shimshilashvili, O.N. Mustafaev, I.V. GoldenkovaPavlova, 2012, published in Genetika, 2012, Vol. 48, No. 11, pp. 1245–1259. REVIEWS AND THEORETICAL ARTICLES

Expression of Heterologous Genes in Plant Systems: New Possibilities A. O. Vyacheslavovaa, I. N. Berdichevetsa, A. A. Tyurina, b, Kh. R. Shimshilashvilia, O. N. Mustafaeva, and I. V. GoldenkovaPavlovaa, b a Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, 119991 Russia email: [email protected] b Russian State Agrarian University, Moscow Timiryazev Agricultural Academy, Moscow, 127550 Russia Received April 16, 2012

Abstract—The basic methods used in current practice for stable and transient expression of heterologous genes in plants are presented and compared. The key areas of research in the heterologous expression of genes in plants have been identified by analyzing literature and experimental data: modeling of metabolic pathways; creation of markerfree transgenic plants; the search for new regulatory elements and plant genes influencing the efficiency of expression of heterologous genes in plants; development of new methods for analyzing of transgenic plants and new approaches to the expression of heterologous genes in plants. DOI: 10.1134/S1022795412110130

BASIC METHODS FOR EXPRESSION interaction with a plant cell, Agrobacterium delivers OF HETEROLOGOUS GENES IN PLANTS part of the plasmid (Ti or Ri plasmids for A. tumefa Two basic strategies are used for the expression of ciens or A. rhizogenes, respectively), a socalled heterologous genes in plants: stable (continuous) TDNA region, into the plant cell with its subsequent expression and transient (temporary) expression. integration into the host plant chromosome. The agro Nuclear or plastid transformation is presently used for bacterial and plant components are both involved in stable expression of heterologous genes in plants. the process of TDNA transfer and integration [6]. Transient expression is mediated by agroinfiltration, Upon interaction with Agrobacterium, the plant cell transformation with viral vectors, and magnifection releases specific sugars and phenols that interact with (Agrobacteriummediated transfection). the receptors on the bacterial cell membrane. A signal from the receptors activates, through a number of mediators, the agrobacterial vir genes located on the Ti Stable Expression plasmid. The proteins encoded in the sequences of the Nuclear transformation. At present, the following vir genes participate in the formation of single main approaches are used for nuclear transformation: stranded TDNA, its attachment to the plant cell wall, (a) agrobacterial transformation; (b) bioballistic trans and its transfer across the agrobacterial and plant cell formation; (c) protoplast transformation; (d) elec membranes into the plant cell. The complex of troporation. TDNA with the Vir proteins is transported in the Agrobacterial transformation is now one of the most cytoplasm and enters the plant cell nucleus. In the widely applied methods to transfer foreign genetic nucleus, the Vir proteins are removed from the material into plants [1]. The soil phytopathogenic TDNA, and the TDNA integrates into the plant cell gramnegative bacterium Agrobacterium tumefaciens is genome with the involvement of plant proteins (Fig. 1). an excellent natural “genetic engineer” and a system for the transfer of heterologous genes and their expres The TDNA region includes several key genetic sion in plants. Due to numerous studies in recent elements that mediate TDNA integration (direct years, highly efficient Agrobacterium strains have been repetitive sequences), selection of transformed plant obtained, new expression vectors have been created, cells in the presence of a selective agent in the nutrient and protocols for transformation in different plant medium (a selective marker gene whose expression is species have been worked out. This has extended the controlled by the promoter), and target gene expres spectrum of host plants, including representatives of sion. Due to the unique characteristic of plant cells, many species of higher plants, both dicotyledonous socalled totipotency, primary plant transformants can and monocotyledonous ones [2–4]. be produced from transformed cells. After molecular, The molecular mechanisms of agrobacterial trans physiological, and biochemical analyses, true trans formation are described in detail in review [5]. After genic plants expressing a target gene with the forma

1067 1068 VYACHESLAVOVA et al.

Agrobacterium

vir genes C h v S E u g a Ti plasmid r TDNA VirD2 s TDNA V i r A V i r P VirE2 G h en

TDNA o VirD1/VirD2 l complex s

wounding

integrated integrated TDNA TDNA

nucleus plant cell

Fig. 1. A diagram of agrobacterial transformation of a plant cell. tion of a protein product are selected from a large protein. Another problem is silencing of target genes number of primary transformants. in transgenic plants. Several factors are known that influence the effi Bioballistic transformation of plants is based on the ciency of agrobacterial transformation of plants. physical process of TDNA delivery. TDNA con These are plant genotype, the explants used, the Agro tained in a plant vector is precipitated onto special bacterium strain, composition of nutrient media, con metal particles (gold, tungsten), and these particles are ditions and duration of the interaction of Agrobacte transferred into the plant genome with the use of a rium and plant explants (inoculation and cocultivation gene gun (bombardment of plant tissue). Thus, the stages), etc. [7]. genes contained in the TDNA region are integrated The method of Agrobacteriummediated transfor into the plant genome and expressed in the trans mation of plants has a number of advantages, includ formed plant cells. The procedures for selecting trans ing a low number of copies of integrated sequences, formed cells and producing from them primary plant the possibility of transferring largesize sequences, the transformants follow the same principle as in the case possibility of expression of genes encoding multimeric of plant transformation with A. tumefaciens. At proteins (e.g., immunoglobulins), and a high level of present, transgenic plants belonging to different fami stable expression (Table). However, despite the appar lies of the plant kingdom have been obtained with this ent simplicity of the method, its main limitation is the method [8]. Since the time this method was developed dependence of transformation efficiency on plant in 1990, numerous bioballistic devices have also genotype. In connection with this, the method often appeared. Different principles of operation and spe requires optimization for an individual plant species or cific design features of these devices necessitate opti vector construct. Taking into consideration the use of mization of the conditions of ballistic transformation transgenic plants obtained by agrobacterial transfor for every device and every plant species [8]. mation as highly efficient producers of target proteins, The efficiency of bioballistic transformation a drawback of this method is an insufficiently high depends on different factors, which are divided into yield of a target protein relative to the total soluble three groups: physical, chemical, and biological. The

RUSSIAN JOURNAL OF GENETICS Vol. 48 No. 11 2012 EXPRESSION OF HETEROLOGOUS GENES IN PLANT SYSTEMS 1069 tion agroinfiltration viral infection Plastid bioballistic transformation transformation of the total soluble protein. protoplast transformation Low copy number Single copy? integration Without integra Without Stable expression expression Transient e into the environment. ed Not required Required required Not Not required bioballistic transformation the physiological maximum >50% integrations and re arrangements Nuclear transformation t proteins can be produced under controlled conditions. e release of a transgen agrobacterial transformation PossibleLow copy number Multiple TDNA PossiblePossible Possible Possible Impossible Possible Possible Impossible Possible Possible Possible , the pollen is free of plastids. Characteristic cy for some species, but special protocols are required. * High transformation efficien cy for some ** Usually from several percent of the total soluble protein to several ** Usually from *** Special approaches are required to prevent th *** Special approaches are required to prevent **** Maternal inheritance of plastids Dependence on genotypeHost plant range DependentVectors to use linear DNAPossibility dependent Less of genes with a size Transfer No 1000 Kb Wideof over DependentCopy number (number of insertions) Less dependent Complex modifications Posttranslational Wide Yesof protein products DependentExpression level Simple efficiencyTransformation Dependent Limited equipmentSpecific expensive Yes High* of proceduresComplexity required Not Simple MediumBiosafety Limited Requir Simple Yes Medium Complex Low Limited Simple Low Complex No Relative*** Low Limited Complex Complex Relative*** low Very No High** Complex Relative*** High High**** Simple High High***** High Simple High High***** Comparative characteristics of stable and transient expression genes in plants Comparative ***** Transgenic plants are not generated. Most recombinan ***** Transgenic

RUSSIAN JOURNAL OF GENETICS Vol. 48 No. 11 2012 1070 VYACHESLAVOVA et al. physical parameters to be experimentally selected are The advantages of this method are the possibility of as follows: helium pressure required to accelerate delivering large DNA fragments and using simple vec DNAcarrying microparticles; vacuum pressure in a tors (in the linear form also) (Table), and the lack of chamber with target cells; distance separating a target agrobacterial contamination during production of tissue from the source of particles and the dissecting transgenic plants. However, protoplast transformation filter; the size and number of microparticles used in a is a rather laborious method with a low transformation shot; the number of shots at a target tissue; etc. The efficiency as compared, for instance, to agrobacterial chemical parameters include the availability and con transformation, and the efficiency of transformation centration of reagents for DNA precipitation on metal depends on the plant genotype (table). All these fac particles, namely, inorganic salts of calcium and mag tors limit the application of protoplast transformation. nesium, and organic components (PEG, glycerol, Electroporation is based on integration of the vector ethanol, spermidine). This group of parameters also TDNA sequence into protoplasts with the use of a includes the pH value, concentration of integrated special device, an electroporator. As a result of physi DNA, and the nature of the metal for microparticles cal treatment, TDNA is transferred into plant cells. (tungsten, gold, platinum). The biological parameters The procedures for selecting transformed cells and are primarily associated with the degree of compe producing plant transformants follow the same princi tence of transformed cells for regeneration of morpho ple as in the case of protoplast transformation [11]. genic structures and subsequent regeneration of Like in the case of direct transfer of DNA into pro plants, with the degree of plasmolysis of explant cells toplasts, the efficiency of electroporation, as a method induced by pre and postosmotic treatment (cultiva for generating transgenic plants, to a large extent tion on a medium with an increased osmotic pres depends on the plant genotype. The method itself is sure), with the nature of integrated DNA and its struc characterized by a low transformation efficiency and ture (doublestranded or singlestranded circular requires specific expensive equipment (table), which DNA, linear doublestranded or singlestranded plas limits its application. mid DNA). A very important factor for the efficiency Plastid transformation is based on the transfer of of bioballistic transformation is the state of the plant genes into chloroplast DNA. A schematic diagram of cell population at the moment of attack, i.e., the ratio plastid transformation is presented in Fig. 3. Only bal of cells at the stage of DNA synthesis and cell division. listic transformation is used for transformation of plas However, despite a seemingly difficult choice of con tids. The procedures for selecting transformed cells ditions for optimal bioballistic transformation, such and producing transformed plants follow the same conditions have already been determined for a large principle as in the case of agrobacterial transformation number of plant species [9]. of plants. The vector DNA is transferred into the plas The main disadvantage of bioballistic transforma tid genome via homologous recombination (Fig. 3). tion, in addition to expensive equipment and the The vector for plastid transformation includes necessity of selecting conditions, is the integration of a sequences homologous to the sequences of the plastid large number of copies of the transgene sequence into DNA genes. This facilitates the integration of the the recipient plant genome, which can lead to trans sequences without disturbing the integrity and func gene silencing (Table). However, it was demonstrated tions of the plastid genome genes. for corn that the number of transformation events with The first successful transformation of plastids was singlecopy integration can be increased using, for performed for the unicellular green alga Chlamydomo example, low DNA quantities for bombardment, but nas reinhardtii and later for tobacco plants [13]. The this can result in a reduction in transformation effi spectrum of plants used for plastid transformation was ciency [10]. It should be noted that in comparison to restricted for a long time to representatives of one or agrobacterial transformation, bioballistics is charac two species. However, technologies for efficient trans terized by a lower transformation efficiency (Table), a formation and generation of transplastomic plants, high probability of integration of sequences of the vec including potato [14], lettuce [15], tomatoes [16], and tor construct, and by the loss of integrity of the expres Arabidopsis [17], are being actively developed at sion cassette [8]. present. The advantages and disadvantages of this method Protoplast transformation (direct transfer of DNA are described in detail in review [13] and are summa into protoplasts) is based on the use of protoplasts of rized in the table. Important advantages are a high tar mesophyll cells (plant mesophyll cells deprived of the get protein yield and the presence of a chaperone cell wall as a result of treatment with a specific set of machine for correct folding of proteins (e.g., human cellulolytic enzymes) [11]. TDNA transfer occurs as serum albumin or interferons); in addition, since a result of alteration of the osmotic pressure inside chloroplasts are prokaryotic in nature, they can plant cells during their treatment with PEG [12]. A express native bacterial genes. Another unquestion schematic diagram for generating transgenic plants able merit of transplastomic plants is their biosafety. with protoplast transformation is presented in Fig. 2 Since the plant pollen does not contain plastids, dis for stable tobacco transformants. persion of transgenes in the environment is impossi

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Creation Isolation of tobacco of an expression vector mesophyll protoplasts

Direct transformation of tobacco protoplasts with PEG

KT

Formation of cell colonies on a selective medium

Callus formation and morphogenesis

Rooting on a selective medium

Molecular genetic analysis

T0 T K

Transplantation into soil, seed production

T1 Segregation analysis

T Analysis of stable transgene 2 heritability in generations

Morphological and biochemical T KT 3 characteristics

Fig. 2. A schematic diagram of generation of transgenic plants with the use of protoplast transformation. K, control; T, transgene.

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rbcL PSGТ P TG Т accD

rbcL accD Chloroplast DNA Homologous recombination

rbcL P SG ТТP TG accD Chloroplast DNA

Fig. 3. A schematic diagram of plastid transformation. rbcL, gene of the large subunit of ribulosebisphosphatecarboxylase/oxy genase; accD, carboxytransferase βsubunit gene; P, promoter; T, terminator; SG, selective marker gene; TG, target gene. ble. Nevertheless, this method has several serious This process is assumed to involve both the agrobacte drawbacks: plastids do not maintain many posttransla rial and plant proteins. tional modifications of proteins, such as glycosylation; Transient expression with the use of viruses is based a number of heterologous proteins in plastids form on the knowledge of viral genomes. Viruses, including inclusion bodies that require repeated folding during plant viruses, have similar genome structures and carry the purification process. Longterm investigations and three main sequences: a sequence encoding the repli optimization of the technology for an individual case protein (involved in replication of viruses in the plant/construct is needed. host organism); a sequence encoding the transport protein (involved in the spread of the virus in the host organism), and a sequence encoding the viral envelope Transient Expression protein (involved in the formation of viral particles). Transient expression of foreign genes in plants is a Figure 4 presents a diagram showing the application of method based on temporary expression of a target viruses for transient expression of target genes and gene in specific plant organs [18]. At present, the fol sequences. In most cases, researchers use the follow lowing main approaches are employed for transient ing approaches: substitution of a target sequence for either the gene encoding the transport protein or the expression of genes in plants: Agrobacteriummediated transformation, expression with the use of plant viral gene encoding the viral envelope protein; insertion of vectors [19], and magnifection. a target sequence between the sequences of the viral genome; insertion of an epitope (an antigen used to Agrobacteriummediated transient expression. As produce vaccines) as a part of the viral envelope pro mentioned above, the process of agrobacterial trans tein (Fig. 4). In the last case, the epitope will be deliv formation can be divided into four steps: attachment ered on the surface of a viral particle. of the bacterium to the plant cell wall, penetration of Magnifection (Agrobacteriummediated transfec TDNA into the plant cell, integration of TDNA into tion) is based on a combination of two methods of the plant genome, and expression of genes from the transient expression (Icon Genetics). The process TDNA region (Fig. 1). In contrast to stable transfor includes vacuum infiltration of whole adult organisms mation, no integration of the transgene into the plant with dilute suspensions of agrobacteria carrying genome occurs in this case, and its expression takes TDNAs encoding RNA replicons. In this case, the place directly from the intact DNA of the agrobacte agrobacteria mediate primary infection and move rium through the system of plant cell replication. ment in cells, while the viral system mediates the The proposed molecular mechanism of transient shortdistance celltocell movement, amplification, expression of genes in plants is described in detail in and a highlevel expression [20]. review [19]. It was shown, in particular, that after exci In general, transient expression of genes is charac sion, the TDNA region exists in the bacterial cell as a terized by a number of advantages (table): a high effi complex of singlestranded DNA (ssTDNA) with the ciency of gene expression and a high level of a target bacterial proteins (T complex). The bacterial proteins protein; only several days are required after infiltration not only stabilize the structure of the T complex, they with agrobacteria or viral particles to obtain a maxi are also involved in its transport to the host plant cell. mum target protein yield (3–4 days for agrobacterial For doublestranded TDNA to be formed, ssTDNA transformation and 10 days for viral infection). Disad is released from the proteins of the mature T complex. vantages include the complexity of the agroinfiltration

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Virus structure Replicase Transport protein Envelope protein

Gene substitution Replicase Target gene Envelope protein

Gene insertion Replicase Transport protein Target gene Envelope protein

Substitution of a gene region Replicase Transport protein TG Envelope protein

Fig. 4. A diagram of transient expression of target genes and sequences with the use of viruses. TG, target gene. technology on an industrilal scale and a low viral vec enzyme catalyzing it. It is also possible to inhibit the tor capacity; i.e., integration and expression of large activity of an enzyme responsible for the delivery of size gene sequences are limited. the substrate or, finally, to manipulate the regulatory The key advantages of transient expression of het factors. erologous genes in plants are a high level of expression As compared to directional selection, genetic engi of target genes; posttranslational modifications of neering permits a desirable trait to be imparted to a plant proteins; the yield of a target protein can reach plant much more rapidly due to modifications of 80% relative to the total protein. Despite the above metabolism of certain compounds. mentioned merits, this method has several drawbacks. The protein products of some genes are toxic for plants We shall present several examples of successful (e.g,. hepatitis B surface antigen); a problem is the modeling of metabolic pathways in plants as a result of expression of oligomultimeric peptides (such as IgG transfer of one or several genes. antibodies), which requires manipulations with viral One of such examples is modeling of metabolism of vectors for the expression of two and more polypep steroid compounds using heterologous expression of tides in equimolar quantities. one gene (CYP11A1 cDNA of cytochrome P450scc from the bovine adrenal cortex) in transgenic tobacco plants [23]. Cytochrome P450scc is known to catalyze CURRENT AREAS OF RESEARCH the ratelimiting step of biosynthesis of mammalian, ON THE EXPRESSION OF HETEROLOGOUS as well as human, steroid hormones. The recent liter GENES IN PLANTS AND PROSPECTS ature data suggest structural and functional conserva OF DEVELOPMENT tion of the steroidogenic and steroid regulatory sys Modeling of Plant Metabolism Through Expression tems in plants and animals. On this theoretical basis of One or Several Heterologous Genes animal CYP11A1 cDNA was chosen for being trans At present, heterologous expression of one gene is ferred and expressed in transgenic tobacco plants. The predominantly used to improve the characteristics of analysis of the obtained transgenic plants showed that agricultural plants. However, a useful trait or a useful the protein product of animal CYP11A1 cDNA is com physiological function in plants is, as a rule, a conse patible with the electrontransport proteins of the quence of the realization of a complex metabolic pathway plants, it displays a specific functional activity and is involving the products of several genes. In connection able to integrate into the steroidogenic system of with this, one of the areas in plant genetic engineering is plants. The expression of this heterologous gene targeted plant metabolism modeling [21]. caused an alteration in the metabolism of steroid com pounds and the formation of metabolites that are not In order to directionally modify the metabolic specific for tobacco plants, such as pregnenolone and pathway, data on metabolomics, transcriptomics, and progesterone. proteomics are necessary for elucidating the key stages of metabolism and possible mechanisms of its regula There are many other examples of successful mod tion. However, plant metabolism is as yet poorly eling of biosynthesis of secondary metabolites in understood and complete information about enzymes plants: an increase in the level of phenolic antioxidants and genes encoding them is known only for individual in potato tubers simultaneously associated with biosynthetic pathways in a number of crops. enhanced expression of the StMtf1M gene and silenc There are two basic approaches to modify the met ing of the F3'5'h gene [24]; alteration of the caffeine abolic pathway in plants: (1) alteration of the meta level in coffee plants [25]; modeling of tropane alka bolic pathway in a desired direction; (2) introduction loid biosynthesis in various plant species via the trans of a new biosynthetic activity from another organism fer of one or two genes [26]. [22]. For instance, the biosynthesis of a particular Modeling of metabolic pathways in plants via product can be stimulated by regulating the ratelim expression of one or several genes is primarily aimed at iting stage of the process via alteraton of the level of an creating plants that produce biologically active sub enzyme catalyzing this reaction. The key problem is stances or to improve the nutritional quality of agricul associated with the knowledge of this stage and the tural crops (biofortification).

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An example of creating plant producers of biologi MarkerFree Transgenic Plants cally active substances are studies on increasing the production of rosmarinic acid (RA) in plants. The It should be noted that the problems in relation to interest of researchers in creating new producers of RA genetically engineered crops are associated with the is explained, first of all, by the range of its biological transfer and expression of selective marker genes action: RA possesses antibacterial, antioxidant, and together with target genes. antiviral properties [27], which makes it a potentially useful compound for the pharmaceutical and cosmet Several selective genes are currently used to provide ics industries. Xiao et al. [28] showed that the level of resistance of transformed cells to antibiotics or herbi RA and its important derivative, lithospermic acid, cides [36]. The employment of selective genes in the can be enhanced by increasing the expression of either transformation process considerably simplifies selec one of the native plant genes encoding the RA biosyn tion among a large number of primary transformants thesis enzymes (c4h, tat, hppr) or two of these genes of exactly those plants that contain the transferred simultaneously (tat and hppr), as well as by suppression genes. Taking into account the public concern about of the native hppd gene. Thus, an approach has been problems of the biosafety of genetically modified proposed for the first time for commercial production plants, such as the probability of transfer of herbicide of these biologically active substances using S. miltior resistance genes to wild relatives of genetically modi rhiza hairy root cultures as producers of RA [28]. fied plants and antibioticresistance genes to soil bac teria or to human gastrointestinal bacteria, researchers As mentioned above, the research on modeling are aiming to create markerfree transgenic plants. metabolic pathways is also aimed at improving the nutritional quality of crops (biofortification). The Several approaches have been proposed for this results of this research can be exemplified by works purpose. The first is to remove the sequence of a selec dealing with the global problem of elimination of vita tive gene or to “switch off” its expression after selec min A deficiency in the populations of the developing tion of primary plant transformants. The selective countries by creating crops with an enhanced content marker gene sequence can be removed using several of carotenoids [29]. Since the time of the pioneer approaches: (a) cotransformation (transformation works on creating “golden rice,” considerable progress with two vectors: one with a target gene and the second has been made in modeling carotenoid metabolism, with a selective marker gene); (b) the use of trans with the use of the genetic engineering methods as posons (Ac/Ds system); (c) the use of an alternative to well. The attention of researchers has also been drawn transposons, namely, the MAT (multiautotransfor to other plant species, such as wheat [30], rape [31], mation) vector system, or (d) sitespecific recombina carrot [32], and tomatoes [33]. In these works, an tion (CreloxP system). The expression of selective approach based on heterologous expression of one or marker genes can be switched off using inducible pro several genes was used. The possibility of simultaneous modeling of three metabolic pathways in plants has moters that drive the expression of selective genes only been demonstrated at present: transgenic corn with an at the stage of selection of primary transformants [37]. increased content in the endosperm of three vitamins Second, the selection of primary transformants can be (βcarotene, ascorbic and folic acids) has been pro made using genes whose products have no negative duced, which is another example of biofortification of influence on plant cells, such as manA or xylA [37], as crops [21]. well as using abiotic stress genes [37]. To model and reconstruct metabolic pathways, a However, all of the described approaches are technology is now being developed for engineering employed (work) only for a small number of plants. In plant minichromosomes, which permits several genes this connection, studies are now underway to search to be transferred at once [34]. With this technology, for new selective genes encoding products that are not several genes can be introduced onto one artificial associated with resistance to antibiotics or herbicides. chromosome; these genes form an individual locus and are very likely to be inherited together and inde In particular, an approach has been developed and pendently of other genes. The use of minichromo tested recently that does not require the use of selective somes opens up considerable possibilities in modeling genes at all and permits direct screening of primary individual metabolic pathways in plants for improving transformants for the product of target gene expres their useful properties, for their protection against sion. Owing to this method, transgenic tobacco plants biotic and abiotic environmental factors, and for pro expressing the gene of the antimicrobial peptide ducing various useful substances, including pharma cecropin P1, as well as tobacco and tomato plants cologically important proteins and peptides. expressing the gene of the hepatitis B virus surface antigen, were obtained and selected [38, 39]. Reconstruction of the metabolic pathways in tobacco Nicotiana ssp. plants [35] is possible not only The progress towards the development of marker through stable expression of heterologous genes, but free plants is described in detail in review [37] basis on also through transient expression of genes in planta. an analysis of experimental articles.

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Expression Vectors for Expression of Heterologous Regulatory Elements and Plant Genes Involved Genes in Plants in the Regulation of Expression of Heterologous Genes in Plants In studies on genetic engineering of plants, the most often used are genetic constructs based on binary It should be noted that the efficiency of expression vectors [40]. At present, a wide range of expression of transferred genes is largely dependent on the design of expression vectors used for plant transformation, vectors has been proposed and applied for plant trans primarily on the regulatory elements controlling the formation. The main ways of modifying plant expres expression of transferred genes. The expression of sion vectors involve introduction of unique restriction transferred genes (transgenes) in plants is controlled at sites (including rare ones) into the polylinker region different stages: transcription, translation, and protein for convenience of target gene cloning; enhancement product stability. The key regulatory elements of expres of the capacity of vectors for cloning largesize (over sion of transferred genes are promoters and enhancers 2000 bp) genes; alteration of the replication point for at the transcriptional level; 5' and 3'untranslated increasing the vector copy number in E. coli and Agro regions of genes at the translational level; leader sig bacterium strains; extension of the spectrum of selec nals and other sequences required for compartmental tive genes for selection of plant transformants; expan ization of protein products and their protection from sion of the application scope, for example, in studies proteolytic degradaton at the level of protein product associated with gene silencing, etc. stability. Below is a short list of the most commonly used In recent years, the efforts of researchers have been aimed at searching for new efficient promoters and, binary vectors: pBin19 [41] and pBI121 vectors [42]; a moreover, at discovering motifs in the promoter series of pCAMBIA vectors with pEarleyGate modifica sequences and elucidating their functional signifi tions [43]; a series of pGreen vectors [44]; GATEWAY cance in determining the level of gene expression. vectors [45]; a series of pBIBAC [46] and pYLTAC During the last five years, more than 100 works have vectors [47, 48]. been published devoted to cloning regulatory At present, the transfer of target gene sequences is sequences from genomes of different plant species and also done with the use of recombination systems to analyzing their promoter activity. This points to the (cre/loxP, GATEWAY) that simplify the cloning pro great interest in searching for and using new regulatory elements able to provide a high level of target gene cess. expression in plants [51, 52]. It should be noted that in Using RMDAP (Recombinationassisted Multi such studies a complex approach based on the combi functional DNA Assembly Platform) technology, nation of bioinformatic analysis of sequences and which incorporates three recombination systems experimental verification of data was most frequently (Gateway technology, in vivo Cre/loxP system, and used, e.g., analysis of regulatory sequences with the recombinant engineering), the possibility of transfer use of reporter systems [53]. ring several genes into plants and their efficient The interest in searching for new promoters is pri expression has been demonstrated recently [49]. A marily explained by the fact that researchers use a series of convenient modular vectors has been con small number of promoters (mainly constitutive) for structed on the basis of this technology for expression the expression of heterologous genes in plants. These or silencing of genes and for creating markerfree promoters include the CaMV 35S RNA promoter transgenic plants [49]. (cauliflower mosaic virus 35S RNA promoter) and the promoters of the ubiquitin and actin genes from differ The choice of a vector for creating experimental ent plant species [53]. plant models is determined by the task of the study and Of special interest nowdays is the search for and depends, for instance, on agrobacterial strains and application of tissue and organspecific promoters, plant species used. No less important for the choice of i.e., promoters providing the expression of a target a plant transformation vector are the size and nature of gene in specific tissues and organs in plants. The most a transferred DNA fragment. For example, in transfer frequently used tissuespecific and organspecific pro ring a sequence of more than 15 Kb in size, it is more moters are the promoters of the potato patatin gene preferable to use vectors of the BIBAC [46] or TAC (expressed predominantly in tuber tissues), corn ole [47] series, which were specially designed for this pur ozin gene (expressed predominantly in seed tissues), pose. and the gene encoding the large subunit of ribulose bis phosphate carboxylase/oxygenase (RBPC) (expressed Despite the fact that it is possible at present to predominantly in leaf tissues) [54]. An example of suc choose a vector for solving a specific research task, cessful application of the organspecific RBPC pro active studies are carried out to develop new genetic moter is the generation of transgenic potato plants constructs for efficient expression of heterologous resistant to Colorado beetle larvae [54]. The use of this genes in plants [50]. promoter permitted the production of potato plants in

RUSSIAN JOURNAL OF GENETICS Vol. 48 No. 11 2012 1076 VYACHESLAVOVA et al. which the target gene, whose product determines protein product levels. To this end, researchers use resistance, is expressed exclusively in plant leaves that such classical methods as Southern, nothern, and are target organs for Colorado beetle larvae. western blot hybridizations, different variants of PCR Alternatives to constitutive and tissuespecific pro analysis, and develop and employ new approaches. An moters are inducible promoters. The use of such pro example of development of a new approach for effi moters permits the control of transgene expression, cient analysis and selection of primary transformants which expands the scope of their application. At is given below. present, inducible expression of transgenes is widely It is known that the integration of a transgene into used both in fundamental studies and in plant biotech the recipient plant genome is a rather rare event. nology to create transgenic crops and plant producers Therefore, vectors with selective genes providing the [55]. The most promising field for the application of tolerance of transformants to selective agents are used chemically regulated (inducible) promoters is the for transformation. The presence of a selective agent in commercial production of recombinant proteins and the culture medium increases the efficiency of selec biofuel with the use of plant cells or green algal cell tion of putative transfromants and considerably cultures [55]. reduces the amount of work. However, since the selec Proteolytic degradation is known to be a serious tion pressure negatively affects the processes of callus barrier for efficient production of heterologous pro formation and morphogenesis in many plant species, teins in plants both in vivo during their expression and it is often needed either to use lower concentrations of in vitro during their isolation from plant tissues. The a selective agent or not to use it at all at early stages. efforts of researchers are currently focused on the This may lead to the selection of false transformants development of strategies for protecting recombinant capable of growing on selective media. PCR with proteins synthesized in plants. Different approaches primers complementary to the sequences of selective are employed for this purpose. One of them is based on genes makes it possible to analyze plant regenerants the use of leader sequences that are able to direct and exclude false transformants from the experiment recombinant proteins to proper plant cell compart already at early stages of their appearance. At present, ments [56]. A correlation between the level of synthe one of the most frequently used methods of plant sis of a recombinant protein and its localization in the transformation is the transfer of genes by agrobacteria. cell has been demonstrated in a series of recent studies It is known that agrobacteria are able to persist in the [57]. Some studies revealed a relationship of certain vascular system of plants within several generations. characteristics of a leader sequence with its functional The presence of agrobacteria in plant specimens may features, i.e., the influence of the nucleotide composi lead to pseudopositive results of PCR, i.e., to the tion, presence of repeats, length, etc., on the localiza selection of false plant transformants. Hence, in order tion of the protein in specific plant organelles. to prove that the sequences of genes under study are It is well known that the level of transgene expres amplified from the plant genomic DNA rather than sion may depend on the site of transgene integration from the expression vector and/or from the agrobacte into the host genome and on the chromatin structure rial genomic DNA, it is necessary to carry out ampli in this region. In the TDNA integration studies per fication with the use of primers selected either for the formed during the last few years with the use of yeast agrobacterial chromosome genes or for the genes and Arabidopsis thaliana as models, plant and bacterial present in the vector construct outside the TDNA proteins involved in the process of TDNA integration region. It is known that the quality of DNA prepara into the host plant genome have been identified. In tions may affect the PCR results. Therefore, some particular, the role of plant histone proteins in agro authors recommend amplifying housekeeping genes as bacterial transformation has been elucidated. It has an internal control [59] to exclude pseudonegative also been found that some histones improve transgene results determined by a low quality of plant genomic expression, protect transgenic DNA being delivered DNA preparations. Thus, regardless of the task facing into the plant cell at the initial stages of transforma a researcher, several PCRs should be performed for tion, and, as a consequence, increase the overall effi efficient selection of true transgenic plants from a large ciency of agrobacterial transformation [58]. number of primary transformants. The conditions for each PCR should be individualized, which eventually takes up much time. To overcome the difficulties in Methods for Analysis of Transgenic Plants selecting primary transformants, a new approach has and New Approaches to the Expression been proposed recently based on the development of a of Heterologous Genes in Plants system of primers, their ratios, and multiplex PCR An important step in studying transgenic plants is conditions, i. e., on the possibility to carry out PCR in their analysis by the methods of molecular biology. It a single tube and receive answers to all the abovemen includes an efficient selection of true transgenic plants tioned questions [60]. among a great number of primary transformants; con As has already been mentioned above, different firmation of the integration of sequences into the plant PCR variants are mainly used to identify transgenes. genome; assessment of expression at the mRNA and They permit the detection of a heterologous sequence

RUSSIAN JOURNAL OF GENETICS Vol. 48 No. 11 2012 EXPRESSION OF HETEROLOGOUS GENES IN PLANT SYSTEMS 1077 in genomic DNA of plants and assessment of the level cokinetics of therapeutic proteins [64]. In addition, of foreign gene transcription in plant cells. Yet, it other advantages are the potential for peroral applica should be emphasized that in most cases it is the pro tion of untreated or partially treated plant material, tein product of a transferred gene that determines new stability of recombinant proteins produced in plants, properties of transgenic plants. It should be added that minimization of the regulatory barriers with respect to most of the products of cloned genes either have no the risk of introduction of plant recombinant proteins enzymatic activity or their enzymatic activity can be into food products, and the absence of ethical prob established with the use of very sophisticated methods. lems that inevitably occur when dealing with trans Researchers continue an active search for new genic animals [64]. approaches to the expression of heterologous genes, At present, the range of plant systems for the one of which is briefly described below. expression of recombinant proteins is very diverse: A new approach has recently been proposed to lines of stable transgenic plants synthesizing target construct experimental models for creating transgenic proteins in different organs and tissues; plant cell cul plants promising for biotechnology. This approach is tures; viral and agrobacterial systems of transient based on the construction and transfer of hybrid genes expression; etc. in which the target gene sequence is transcriptionaly It should be noted that the list of host plants for the and translationally fused to the sequence of the production of pharmacological proteins is being con reporter gene encoding thermostable lichenase. This stantly extended [65]. It includes (but is not restricted method was successfully tested in constructing and to) Nicotiana ssp., Arabidopsis thaliana, lucern, spinach, expressing hybrid genes that contained the following potato, duckweed, strawberry, carrot, tomatoes, aloe, as target genes: recA gene, whose product participates and unicellular algae. Various proteins are expressed in in recombination processes [61]; cry3aM gene encod corn, rice, bean, and tobacco seeds; in potato, tomatoes, ing deltaendotoxin, which provides plant resistance and strawberry; in tobacco and corn suspension cell cul to Colorado beetle larvae [54]; desA gene encoding tures; in hairy root cultures; and in transformed chloro delta12desaturase, which provides plant resistance plasts of different plant species [65, 66]. to abiotic and biotic factors [62]; genes encoding Thus, much knowledge has been accumulated at spidroin 1 (spider dragline silk protein), which is char present about the key mechanisms of transfer of heter acterized by unique properties [63]. The results ologous genes and their expression in plants. A large obtained demonstrate that with this approach, the body of methodological material is also available for level of expression of a target gene can be assessed by stable and transient expression of genes in plants. simple and sensitive methods at the level of formation However, it is still not completely understood what of its protein product; consequently, this approach factors, including genetic determinants, ensure highly enables optimal systems for expression of heterolo efficient expression of heterologous genes in plants. gous genes in plants to be elaborated and optimal experimental models of transgenic plants that are On the one hand, the vast amount of information promising for fundamental research and biotechnol on plant genes and genomes and on the level of gene ogy to be desinged. expression in plants obtained with the use of upto date microarray technologies should be subjected to the bioinformatic analysis, it is necessary to carry out Expression of Heterologous Genes in Plants searching for genetic determinants that are essential for the Production of Important for a highly efficient expression of heterologous genes Pharmacological Proteins in plants at all levels of expression regulation: tran Studies have been carried out for nearly twenty scription, posttranslational modifications, translation, years already on the use of plants as producers of phar and stability of protein products. On the other hand, macological proteins and peptides (biopharming). experimental confirmation of the identified genetic determinants is also required, for which efficient tools, Plants have certain advantages over such expression such as new vector systems, new approaches to the systems as microbes, yeast, or insect system and mam expression of genes, and new methods of analysis malian cell cultures. The results of the past two should be developed. decades show that plant expression systems allow a large quantity of biopreparations to be obtained with low economic costs and free of human viruses, prions REFERENCES and bacterial endotoxins. Since plant cells are known 1. Tzfira, T. and Citovsky, V., AgrobacteriumMediated to possess the system of protein posttranslational Genetic Transformation of Plants: Biology and Bio modification similar to that in animal cells, the basic technology, Curr. Opin. Biotechnol., 2006, vol. 17, advantage of transgenic plants, as compared to other pp. 147–154. systems available for largescale and cheap production 2. Azria, D. and Bhalla, P.L., AgrobacteriumMediated of biopreparations, such as E. coli or yeast, is their Transformation of Australian Rice Varieties and Pro capacity to perform most posttranslational modifica moter Analysis of Major Pollen Allergen Gene Ory S 1, tions required for the biological activity and pharma Plant Cell Rep., 2011, vol. 30, pp. 1673–1681.

RUSSIAN JOURNAL OF GENETICS Vol. 48 No. 11 2012 1078 VYACHESLAVOVA et al.

3. Liu, H., Xie, X., Sun, S., et al., Optimization of Agro nant Proteins in Plants, Recent Pat. Biotechnol., 2008, bacteriumMediated Transformation of Sunflower vol. 2, no. 3, pp. 198–208. (Helianthus annuus L.) Immature Embryos, AJCS, 20. Gleba, Y., Klimyuk, V., and Marillonnet, S., Magnifec 2011, vol. 5, no. 12, pp. 1616–1621. tion—a New Platform for Expressing Recombinant 4. Ziemienowicz, A., Shim, YS., Matsuoka, A., et al., A Vaccines in Plants, Vaccine, 2005, vol. 18, pp. 2042– Novel Method of Transgene Delivery into Triticale 2048. Plants Using the Agrobacterium TDNADerived 21. Naqvia, S., Zhua, C., Farrea, G., et al., Transgenic NanoComplex, Plant Physiol., 2012, vol. 158, no. 4, Multivitamin Corn through Biofortification of pp. 15031513. Endosperm with Three Vitamins Representing Three 5. DafnyYelin, M., Levy, A., and Tzfira, T., The Ongoing Distinct Metabolic Pathways, Proc. Natl. Acad. Sci. Saga of Agrobacterium–Host Interactions, Trends Plant U.S.A., 2009, vol. 106, no. 19, pp. 7762–7767. Sci., 2008, vol. 13, no. 3, pp. 102–105. 22. Davies, K.M., Genetic Modification of Plant Metabo 6. Veena, J.H., Doerge, R.W., and Gelvin, S.B., Transfer lism for Human Health Benefits, Mutat. Res., 2007, of TDNA and Vir Proteins to Plant Cells by Agrobac vol. 622, nos. 1–2, pp. 122–137. terium tumefaciens Induces Expression of Host Genes 23. Spivak, S.G., Berdichevets, I.N., Litvinovskaya, R.P., Involved in Mediating Transformation and Suppresses et al., Some Peculiarities of Steroid Metabolism in Host Defense Gene Expression, Plant J., 2003, vol. 35, Transgenic Nicotiana tabacum Plants Bearing the pp. 219–236. CYP11A1 cDNA of Cytochrome P450SCC from the 7. Opabode, J.T., Agrobacterium Mediated Transforma Bovine Adrenal Cortex, Russ. J. Bioorg. Chem., 2010, tion of Plants: Emerging Factors That Influence Effi vol. 36, no. 2, pp. 224–232. ciency, Biotech. Mol. Biol. Rev., 2006, vol. 1, pp. 12–20. 24. Rommens, C.M., Richael, C.M., Yan, H., et al., Engi 8. Taylor, N.J. and Fauquet, C.M., Microparticle Bom neered Native Pathways for High Kaempferol and Caf bardment as a Tool in Plant Science and Agricultural feoylquinate Production in Potato, Plant Biotechnol. J., Biotechnology, DNA Cell Biol., 2002, vol. 21, pp. 963– 2008, vol. 6, pp. 870–886. 977. 25. Ogita, S., Uefuji, H., Morimoto, M., and Sano, H., 9. Fadeev, V.S., Shimshilashvili, Kh.R., and Gapo Metabolic Engineering of Caffeine Production, Plant nenko, A.K., Induction, Regeneration, and Ballistic Biotechnol., 2005, vol. 22, pp. 461–468. Sensitivities of Different Genotypes of Common Wheat 26. Zhang, L. and Kai, GY., Lu, B.B., et al., Metabolic (Triticum aestivum L), Russ. J. Genet., 2008, vol. 44, Engineering of Tropane Alkaloid Biosynthesis in no. 9, pp. 1096–1104. Plants, J. Integr. Plant Biol., 2005, vol. 47, no. 2, 10. Lowe, B.A., Shiva Prakash, N., Way, M., et al., pp. 136–143. Enhanced Single Copy Integration Events in Corn via 27. Petersen, M., Abdullah, Y., Benner, J., et al., Evolution Particle Bombardment Using Low Quantities of DNA, of Rosmarinic Acid Biosynthesis, Phytochemistry, Transgenic Res., 2009, vol. 18, no. 6, pp. 831–840. 2009, vol. 70, nos. 15–16, pp. 1663–1679. 11. Potrycus, I. and Schillito, R.D., Protoplast: Isolation, 28. Xiao, Y., Zhang, L., Gao, S., et al., The c4h, tat, hppr Culture, Plant Regeneration, Plant. Mol. Biol., 1986, and hppd Genes Prompted Engineering of Rosmarinic vol. 118, pp. 549–578. Acid Biosynthetic Pathway in Salvia miltiorrhiza Hairy Root Cultures, PLoS ONE, 2011, vol. 6, no. 12. 12. Paszkowski, J., Shillito, R.D., and Saul, M., Direct Gene Transfer to Plants, EMBO J., 1984, vol. 3, 29. Bai, C., Twyman, R.M., Farr, G., et al., A Golden pp. 2717–2722. Era—ProVitamin A Enhancement in Diverse Crops, In Vitro Cell. Dev. Biol.: Plant, 2011, vol. 47, pp. 205– 13. Shchelkunov, S.N., Konstantinov, Yu.M., and 221. Deineko, E.V., Transplastomic Plants, Vavilovskii Zh. 30. Cong, L., Wang, C., Chen, L., et al., Expression of Genet. Sel., 2011, vol. 15, no. 4, pp. 808–817. Phytoene Synthase1 and Carotene Desaturase crtI 14. Scotti, N., Valkov, V.T., and Cardi, T., Improvement of Genes Result in an Increase in the Total Carotenoids Plastid Transformation Efficiency in Potato by Using Content in Transgenic Elite Wheat (Triticum aestivum Vectors with Homologous Flanking Sequences, GM L.), J. Agric. Food Chem., 2009, vol. 57, pp. 8652–8660. Crops, 2011, vol. 2, no. 2, pp. 89–91. 31. Fujisawa, M., Takita, E., Harada, H., et al., Pathway 15. Lim, S., Ashida, H., Watanabe, R., et al., Production of Engineering of Brassica napus Seeds Using Multiple Biologically Active Human Thioredoxin 1 Protein in Key Enzyme Genes Involved in Ketocarotenoid For Lettuce Chloroplasts, Plant Mol. Biol., 2011, vol. 76, mation, J. Exp. Bot., 2009, vol. 60, pp. 1319–1332. pp. 335–344. 32. Maass, D., Arango, J., Wust, F., et al., Carotenoid 16. Apel, W. and Bock, R., Enhancement of Carotenoid Crystal Formation in Arabidopsis and Carrot Roots Biosynthesis in Transplastomic Tomatoes by Induced Caused by Increased Phytoene Synthase Protein Lev LycopenetoProvitamin A Conversion, Plant Physiol., els, PLoS ONE, 2009, vol. 4. 2009, vol. 151, pp. 59–66. 33. Fraser, P.D., Enfissi, E.M.A., and Bramley, P.M., 17. Lutz, K.A., Azhagiri, A., and Maliga, P., Transplastom Genetic Engineering of Carotenoid Formation in ics in Arabidopsis: Progress toward Developing an Effi Tomato Fruit and the Potential Application of Systems cient Method, Methods Mol. Biol., 2011, vol. 774, and Synthetic Biology Approaches, Arch. Biochem. pp. 133–47. Biophys., 2009, vol. 483, pp. 196–204. 18. Gleba, Y., Klimyuk, V., and Marillonnet, S., Viral Vec 34. Yu, W., Han, F., and Birchler, J.A., Engineered Min tors for the Expression of Proteins in Plants, Curr. Opin. ichromosomes in Plants, Curr. Opin. Biotechnol., 2007, Biotechnol., 2007, vol. 18, pp. 134–141. vol. 18, pp. 425–431. 19. Sheludko, Y.V., AgrobacteriumMediated Transient 35. Tremblay, R., Wang, D., Jevnikar, A.M., and Ma, S.W., Expression as an Approach to Production of Recombi Tobacco, a Highly Efficient Green Bioreactor for Pro

RUSSIAN JOURNAL OF GENETICS Vol. 48 No. 11 2012 EXPRESSION OF HETEROLOGOUS GENES IN PLANT SYSTEMS 1079

duction of Therapeutic Proteins, Biotechnol. Adv., Genes in Plants, In Vitro Cell. Dev. Biol.: Plant, 2011, 2010, vol. 28, pp. 214–221. vol. 47, pp. 549–550. 36. Barampuram, S. and Zhang, Z.J., Recent Advances in 52. Park, S.H., Yi, N., Kim, Y.S., et al., Analysis of Five Plant Transformation, Plant Chromosome Engineer Novel Putative Constitutive Gene Promoters in Trans ing: Methods and Protocols, Methods Mol. Biol., 2011, genic Rice Plants, J. Exp. Bot., 2010, vol. 61, no. 9, vol. 701, pp. 1–35. pp. 2459–2467. 37. Upadhyaya, C.P., Nookaraju, A., Gururani, M.A., 53. Mann, D.G., King, Z.R., Liu, W., et al., Switchgrass et al., An Update on the Progress Towards the Develop (Panicum virgatum L.) Polyubiquitin Gene (PvUbi1 and ment of MarkerFree Transgenic Plants, Bot. Stud., PvUbi2) Promoters for Use in Plant Transformation, 2010, vol. 51, pp. 277–292. BMC Biotechnol., 2011, vol. 11, no. 11, p. 74. 38. Rukavtsova, E.B., Gayazova, A.R., Chebotareva, E.N., 54. Salehi Jouzani, Gh.R. and Goldenkova, I.V., A New and Buryanov, Ya.I., Production of MarkerFree Plants Reporter Gene Technology: Opportunities and Per Expressing the Gene of the Hepatitis B Virus Surface spectives, Iranian J. Biotechnol., 2005, vol. 3, no. 1, Antigen, Russ. J. Genet., 2009, vol. 45, no. 8, pp. 924– pp. 1–15. 928. 55. Salehi Jouzani, G., Goldenkova, I.V., and Piruzian, 39. Zakharchenko, N.S., Pigoleva, S.V., Yukhmanova, A.A., E.S., Expression of Hybrid cry3aM–licBM2 Genes in and Buryanov, Y.I., Use of the Gene of Antimicrobial Transgenic Potatoes (Solanum tuberusom), J. Plant Cell Peptide Cecropin P1 for Producing MarkerFree Tissue Organ Cult., 2008, vol. 92, pp. 321–325. Transgenic Plants, Russ. J. Genet., 2009, vol. 45, no. 8, 56. Corrado, G. and Karali, M., Inducible Gene Expres pp. 929–933. sion Systems and Plant Biotechnology, Biotechnol. 40. Miki, B. and McHugh, S., Selectable Marker Genes in Adv., 2009, vol. 27, pp. 733–743. Transgenic Plants: Applications, Alternatives and Bio 57. GoldenkovaPavlova, I.V., Mirakhorli, N., Maali, A.R., safety, J. Biotechnol., 2004, vol. 107, pp. 193–232. et al., Experimental Models for Creation of Transgenic 41. Lee, LY. and Gelvin, S.B., TDNA Binary Vectors and Plants Resistant to Stress Factors, Tsitol. Genet., 2007, Systems, Plant Physiol., 2008, vol. 146, pp. 325–332. no. 3, pp. 44–49. 42. Bevan, M.W., Binary Agrobacterium Vectors for Plant 58. Rojo, E. and Denecke, J., What Is Moving in the Secre Transformation, Nucleic Acids Res., 1984, vol. 12, tory Pathway of Plants?, Plant Physiol., 2008, vol. 147, pp. 8711–8721. pp. 1493–1503. 43. Jefferson, R.A., Kavanagh, T.A., and Bevan, M.W., 59. Tenea, G.N., Spantzel, J., Lee, LY., et al., Overex GUS Fusions: 3Glucuronidase as a Sensitive and Ver pression of Several Arabidopsis Histone Genes satile Gene Fusion Marker in Higher Plants, EMBO J., Increases AgrobacteriumMediated Transformation and 1987, vol. 6, pp. 3901–3907. Transgene Expression in Plants, Plant Cell, 2009, 44. Earley, K.W., Haag, J.R., Pontes, O., et al., Gateway vol. 21, pp. 3350–3367. Compatible Vectors for Plant Functional Genomics 60. Mannerlof, M., Tuvesson, S., Steen, P., and Tenning, and Proteomics, Plant J., 2006, vol. 45, no. 4, pp. 616– P., Transgenic Sugar Beet Tolerant to Glyphosate, 629. Euphytica, 1997, vol. 94, pp. 83–91. 45. Hellens, R.P., Edwards, E.A., Leyland, N.R., and 61. Berdichevetz, I.N., Shimshilashvili, C.R., Gerasy Bean, S., Mullineaux PM PGreen: A Versatile and menko, I.M., et al., Multiplex PCR Assay for Detec Flexible Binary Ti Vector for AgrobacteriumMediated tion of Recombinant Genes Encoding Fatty Acid Plant Transformation, Plant Mol. Biol., 2000, vol. 42, Desaturases Fused with Lichenase Reporter Protein in pp. 819–832. GM Plants, Anal. Bioanal. Chem., 2010, vol. 397, no. 6, 46. Karimi, M., Inze, D., and Depicker, A., GATEWAY pp. 2289–2293. Vectors for AgrobacteriumMediated Plant Transforma 62. Komakhin, R.A., Komakhina, V.V., Milyukova, N.A., tion, Trends Plant Sci., 2002, vol. 7, pp. 193–195. et al., Transgenic Tomato Plants Expressing recA and 47. Hamilton, C.M., A BinaryBAC System for Plant NLSrecAlicBM3 Genes as a Model for Studying Mei Transformation with HighMolecular Weight DNA, otic Recombination, Russ. J. Genet., 2010, vol. 46, Gene, 1997, vol. 200, pp. 107–116. no. 12, pp. 1440–1448. 48. Chen, Q.J., Zhou, H.M., Chen, J., and Wang, X.C., A 63. Amiri, R.M. and Yur’eva, N.O., Shimshilashvili, Khr.R., GatewayBased Platform for Multigene Plant Transfor et al., Expression of AcylLipid 12Desaturase Gene in mation, Plant Mol. Biol., 2006, vol. 62, pp. 927–936. Prokaryotic and Eukaryotic Cells and Its Effect on 49. Liu, Y.G., Shirano, Y., Fukaki, H., et al., Complemen Cold Stress Tolerance of Potato, J. Integr. Plant Biol., tation of Plant Mutants with Large Genomic DNA 2010, vol. 52, no. 3, pp. 289–297. Fragments by a TransformationCompetent Artificial 64. Piruzyan, E.S., Bogush, V.G., Sidoruk, K.V., et al., Chromosome Vector Accelerates Positional Cloning, Construction of Synthetic Genes for Analogs of Spider Proc. Natl. Acad. Sci. U.S.A., 1999, vol. 96, pp. 6535– Silk Spidroin 1 and Their Expression in Tobacco 6540. Plants, Mol. Biol., 2003, vol. 37, no. 4, pp. 554–560. 50. Ma, L., Dong, J., Jin, Y., et al., RMDAP: A Versatile, 65. Desai, P.N., Shrivastava, N., and Padh, H., Production ReadytoUse Toolbox for Multigene Genetic Trans of Heterologous Proteins in Plants: Strategies for Opti formation, PLoS ONE, 2011, vol. 6. mal Expression, Biotech. Adv., 2010, vol. 8, no. 4, 51. Vyacheslavova, A.O., Berdichevets, I.N., Shimshilash pp. 427–435. vili, H.R., et al., A Series of Modular Vectors for Clon 66. Rybicki, E.P., PlantProduced Vaccines: Promise and ing of Target Genes and Regulatory Elements for Pro Reality, Drug Discovery Today, 2009, vol. 14, nos. 1–2, viding Stable and Effective Expression of Heterological pp. 16–24.

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View publication stats Nature and Science, 3(1), 2005, Ma and Chen, Gene Transfer Technique

Gene Transfer Technique

Hongbao Ma, Guozhong Chen

Michigan State University, East Lansing, MI 48823, USA, [email protected]

Abstract: This article is describing the nine principle techniques for the gene transfection, which are: (1) lipid-mediated method; (2) calcium-phosphate mediated; (3) DEAE-dextran-mediated; (4) electroporation; (5) biolistics (gene gun); (6) viral vectors; (7) polybrene; (8) laser transfection; (9) gene transfection enhanced by elevated temperature, as the references for the researchers who are interested in this field. [Nature and Science. 2005;3(1):25-31].

Key words: DNA; gene; technique; transfer

agricultural potential and medical importance 1 Introduction (Campbell, 1999; Uzogara, 2000; Lorence, 2004). The gene transfer methods normally include three categories: 1. transfection by biochemical methods; 2. Gene transfer is to transfer a gene from one DNA transfection by physical methods; 3. virus-mediately molecule to another DNA molecule. Gene transfer transduction. The gene transfer results can be transient represents a relatively new possibility for the treatment and stable transfection. of rare genetic disorders and common multifactorial Gene therapy can be defined as the deliberate diseases by changing the expression of a person's genes transfer of DNA for therapeutic purposes. Many (Arat, 2001). In 1928, Griffith reported that a serious diseases such as the tragic mental and physical nonpathogenic pneumoccocus strain could become handicaps caused by some genetic metabolic disorders pathogenic when it was mixed with cells of heat-killed may be healed by gene transfer protocol. Gene transfer pathogenic pneumoccocus, which hinted that the is one of the key factors in gene therapy (Matsui, pathogenic genetic material could be transformed from 2003), and it is one of the key purposes of the clone the heat-killed pathogenic pneumoccocus to the (Ma, 2004). nonpathogenic strain (Griffith, 1928). This is the first Gene transfer can be targeted to somatic (body) or report for gene transfer observation. However, the germ (egg and sperm) cells. In somatic gene transfer transforming substance was not identified in these the recipient's genome is changed, but the change will experiments. Up to 1944, Avery et al demonstrated that not be passed on to the next generation. In germline deoxyribonucleic acid (DNA) was the transforming gene transfer, the parents' egg and sperm cells are substance (Avery, 1944). In 1952, Hershey and Chase changed with the goal of passing on the changes to showed that DNA was the only material transferred their offspring. Germline gene transfer is not being during bacteriophage infection, which suggested that actively investigated, at least in larger animals and the DNA is the genetic material (Hershey, 1952). humans (Bordignon, 2003; Umemoto, 2005). The basic technique for introducing DNA into E. coli have inspired procedures for the introduction of 2 Transient and Stable Transfection DNA into cells from a wide variety of organisms, including mammalian cells. Genetic engineering of 2.1 Transient transfection food is the science which involves deliberate In transient transfection, the transfected DNA is modification of the genetic material of plants or not integrated into host chromosome. DNA is animals. Introduction of DNA into plants is of great transferred into a recipient cell in order to obtain a

http://www.sciencepub.org · 25 · [email protected] Nature and Science, 3(1), 2005, Ma and Chen, Gene Transfer Technique temporary but high level of expression of the target (6) Replace the medium in the cells with 0.2 ml gene. transfection medium without serum. (7) Add 0.15 ml medium without serum to the 2.2 Stable transfection tube containing the complexes for each well. Stable transfection is also called permanent (8) Incubate the cells with the complexes for about o tran sfection. By the stable transfection, the transferred 10 hours at 37 C in a CO2 incubator. The incubating DNA is integrated (inserted) into chromosomal DNA time will be flexible by the cell type. and the genetics of recipient cells is permanent changed. (9) Add 0.4 ml growth medium containing double the 2× normal concentration of the serum without 3 Transfection Methods removing the transfection mixture. (10) Replace the medium with fresh, complete Generally, there are 9 ways for gene transfer: (1) medium at 20 hours following the start of transfection Lipid-mediated method; (2) Calcium-phosphate med- if continued cell growth is required. iated; (3) DEAE-dextran-mediated; (4) Electroporation; (11) Assay cell extracts for transient gene (5) Biolistics; (6) Viral vectors; (7) Polybrene; (8) La- expression at 24-72 hours after transfection, depending ser transfection; (9) Gene transfection enhanced by ele- on the cell type and promoter activity. vated temperature (Sambrook, 2001). (12) To obtain stable transfectants, passage the cells 1:10 into the selective medium after 72 hours of 3.1 Lipid-mediated method transfection for the reporter gene transfected. This method can be used for both transient and stable transfection, and it can be used for adherent cells, 3.2 Calcium-phosphate mediated primary cell lines, and suspension cultures. For the To get a better description, the following protocol following protocol, the Lipofectamine Reagent from is using the human interleukin-2 gene transfer into Invitrogen Corporation will be used. Lipofectamine cultured rat myocytes as the example manual. Reagent is a 3:1 (w/w) liposome formulation of the plycationic lipid 2,3-dioleyloxy-N-[2(sperminecardox- 3.2.1 Rat heart muscle cells are primarily culture- ido)ethyl]-N,N-dimethyl-1-propanaminium trifluoro- ed: acetate (DOSPA) and the neutral lipid dioleoyl (1) Adult rats are sacrificed by decapitation with a phosphatidylethanolamine (DOPE) in membrane- decapitator. filtered water (Catalogue Number 18324, Invitrogen (2) Rat hearts are moved out and left atria are Corporation, Carlsbad, California, USA) (Hawley- isolated under sterile condition. Nelson, 1993; Shih, 1997). (3) Tissue is transferred to a fresh sterile (1) Put about 40,000 cells per well of a 24-well phosphate buffered solution (PBS) and rinse. plate in 0.5 ml of the appropriate complete growth (4) Transfer to a second dish and dissect off medium (add 10% serum if it needs). unwanted tissue such as fat or necrotic material and o (2) Incubate the cells at 37 C in a CO2 incubator chop finely with crossed scalpels to about 1 mm cubes. until the cells are 50-80% confluent (about 20 hours, (5) Transfer by pipette (10 – 20 ml with wide tip) depending on the cells). to a 15-ml sterile centrifuge tube. (3) Dilute 3 µg DNA into 25 µl medium without (6) Wash by resuspending the pieces in PBS, serum for each well and mix. transfer the chopped pieces to the trypsinization flask, (4) Dilute 3 µl Lipofectamine Reagent into 25 µl and add 1 ml trypsin solution (0.25%) per 100 mg medium without serum for each well and mix. tissue. Incubate the tissue in trypsin solution for 12 o (5) Combine diluted DNA (Step 3) and Lipofect- hours at 4 C then wash with PBS for 3 times. amine Reagent (Step 4) and incubate at room (7) Add 1 ml trypsin solution (0.25%) per 100 mg temperature for 30 min. In this step the DNA-liposome tissue, with 1 mg/ml elastase and 1 mg/ml collagenase o complexes are formed. then stir at about 200 rpm for 30 min at 36.5 C.

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(8) Allowing the pieces to settle, collect B. Add 200 µl of freshly prepared Solution II supernatant, centrifuge at approximately 500 g for 5 (0.2 N NaOH, 1% SDS), inverting the tube min, resuspending pellet in 10 ml medium with 10% rapidly 5 times. Do not vortex. Store at 4oC. serum (FBS) (Gibco BRL Life Technologies, Inc., C. Add 150 µl ice-cold Solution III (5 M Grand Island, NY, USA), and store cells on ice. potassium acetate 60 ml, glacial acetic acid

(9) Add fresh trypsin to pieces and continue to stir 11.5 ml, H2O 28.5 ml), on ice for 3-5 min. and incubate for a further 30 min. Repeat steps 6 – 8 D. Centrifuge at 12,000g for 10 min, at 23oC . until complete disaggregation occurs or until no further E. Pour supernatant into QIAprep column disaggregation is apparent. (silicon gel column, Qiagen Company, USA). (10) Collect and pool chilled cell suspensions, and F. Centrifuge at 12000g for 1 min and discard count by hemocytometer. flow through. 6 (11) Dilute to 10 per ml in growth medium and G. Wash the column with 0.75 ml PE buffer (55 seed as many flasks as are required with approximately ml of 5 mM Mops-KOH, pH 7.5-7, 0.75 mM 5 2 x 10 cells per ml or set up a range of concentrations NaCl plus 220 ml of ethanol). from about 10 mg tissue per ml. H. Centrifuge 1 min at 12000g and discard flow (12) Put into CO2 incubator with 36.5oC. through. (13) Culture medium used is Medium 199 with I. Place column in 1.5 ml microcentrifuge tube. 10% FBS. All the solutions used contain 0.1 mg/ml of J. Add 50 µl of the DEPC H2O in the center of anti-biotic ampicillin (Sigma, St Louis, MO, USA). the column, stand for 1 min, centrifuge at 12000g for 1 min. 3.2.2 Bacteria Culture (Sambrook, 1989; Frederick, K. Take 1 µl of DNA (plasmid), add 99 µl of TE 1992): buffer, pH 8.0, measure DNA concentration (1) Growth of E. coli: Dissolve E. coli in 0.3 ml at OD260 nm and OD280 nm (OD260 LB plus tetracycline (2 mg/ml) medium, transfer it into nm/OD280 nm should be >1.7). a tube containing 5 ml LB plus tetracycline (2 mg/ml) L. Redissolve the DNA in 50 µl of TE (pH 8.0) o o medium, 37 C overnight, then freeze it at -70 C. containing DNAase-free pancreatic RNAase (2) Harvesting E. coli: o (20 µg/ml). Vortex briefly. Store at -20 C. A. Streak an inoculum across one side of a plate. M. Calculate the concentration of the plasmid Resterilize an inoculating loop and streak a DNA: 1 OD260 nm = 50 µg of plasmid sample from the first streak across a fresh part DNA/ml. Store the DNA in aliquots at -20oC. of plate, then incubate at 37oC until colonies

appear (overnight). 3.2.3 Transfer human interleukin-2 gene into rat B. Transfer a single bacterial colony into 2 ml of heart muscle cells: LB medium containing tetracycline (2 mg/ml) (1) Transferred gene: Human interleukin-2 (IL- in a loosely capped 15-ml tube. 37oC 2) gene cloned in plasmid pBR322 inserted in E. coli overnight with vigorous shaking. can be bought from American Type Culture Collection C. Pour 1.5 ml of the culture into a microfuge (ATCC, Rockville, MD, USA). tube. Centrifuge at 12,000g for 30 seconds at (2) Transfection: ∼2×107 of heart muscle cells 4oC in a microfuge. Store remainder at 4oC. suspended in 0.2 ml medium are seeded into a tissue D. Remove the medium by aspiration. culture chamber. 48-72 hours later, remove medium (3) Lysis of E. coli and purification of plasmid: and add 0.2 ml fresh medium, then add 0.5 µg of A. Resuspend E. coli pellet in 100 µl of ice-cold plasmid in 0.05 ml calcium phosphate-HEPES-buffered Solution I (50 mM glucose, 25 mM Tris-Cl, saline, pH 7.0, at 37oC. pH 8.0, 10 mM EDTA, pH 8.0).

3.2.4 Detection of interleukin-2:

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12~48 hours after the addition of plasmid and moatic activity in cell extract. incubation, the amount of interleukin-2 is measured with the indirect enzyme-linked immunosorbent assay 3.4 Electroporation (ELISA) in medium. Antibody of anti-interleukin-2 Pulse electrical fields can be used to introduce (human) can be gotten from Sigma (Sigma Chemical DNA into cells of animal, plant and bacteria. Factors Co., St Louis, MO, USA). that influence efficiency of transfection by electroporation: applied electric field strength, electric 3.3 DEAE-dextran mediated pulse length, temperature, DNA conformation, DNA DEAE-dextran (diethylaminoethyloethyl-dextran) concentration, and ionic composition of transfection was used to introduce poliovirus RNA and SV40 and medium, etc. polyomavirus DNAs into cells in 1960s (Pagano, 1965; Steps of the electroporation transfection: McCutchan, 1968; Warden, 1968). (1) Harvest cells in the mid- to late-logarithmic There are three points that DEAE-dextran phase of growth. mediated transfection differs from calcium phosphate (2) Centrifuge at 500 g (2000 rpm) for 5 min at coprecipitation. (1) It is used for transient transfection. 4oC. (2) It works more efficiently with cell lines of BSC-1, (3) Resuspend cells in growth medium at concen- CV-1 and COS, etc. (3) It is more sensitive. tration of 1 X 107 cells/ml. The DEAE-dextran mediated transfection could be (4) Add 20 µg plasmid DNA in 40 µl cells. done by the following steps: (5) Electric transfect by 300 V / 1050 µF for 1-2 (1) Harvest exponentially growing cells by min. trypsinization and transfer then into 60-mm tissue (6) Transfer the electroporated cells to culture dish culture dished at a density of 105 cells/dish. and culture the cells. (2) Add 5 ml complete growth medium. (7) Assay DNA, RNA or protein and continuously o (3) Incubate 24 hours at 37 C with 5% CO2. culture the cells to get positive cell lines. . (4) Prepare DNA/DEAE-dextran/TBS-D solution by mixing 2 mg of superoiled plasmid DNA into 1 3.5 Polybrene µg/ml DEAE-dextran in TBS-D. Several polycations, including polybrene (1,5- (5) Remove medium and wash tree times with dimethyl-1,5-diazaundecamethylene poly- PBS and twice with TBS-D. methobromide) (Chaney, 1986) and poly-L-ornithine (6) Add DNA/DEAE-dextran/TBS-D solution 250 (Nead, 1995), have been used in gene transfection with µl. the DMSO enhancement. Normal steps are following: o (7) Incubate 60 min at 37 C with 5% CO2. (1) Harvest exponential cells by trypsinzationin (8) Remove DNA/DEAE-dextran/TBS-D solution. and replate at a density of 5,000 cells/mm2 in 10 ml (9) Wash with TBS-D three time and PBS twice. MEM-α containing 10% fetal calf serum. o (10) Add 5 ml medium supplemented with serum (2) Incubate 24 hours at 37 C in 5% CO2. and chloroquine (0.1 mM). (3) Replace medium with 3 ml pre-warmed med- o (11) Incubate 4 hours at 37 C with 5% CO2. ium containing serum, 10 µg DNA and 30 µg (12) Remove medium. polybrene (37oC). Mix the medium before adding (13)Wash with serum-free medium three times. polybrene. (14) Add to cells 5 ml of medium supplement with (4) Incubate 12 hours with a gent shake each hour. o serum, and incubate 48 hours at 37 C with 5% CO2. (5) Remove medium and add 5 ml 30%^ DMSO (15) Harvest the cells after the 48 hours transfer- in serum-containing medium. ction. (6) After 4 min incubation, aspirate the DMSO (16) Analyze RNA or DNA by hybridization, or solution. Wash the cells twice with warmed (37oC) analyze expressed protein by radiommunoassay, serum-free medium, and add 10 ml complete medium immunoblotting, immuniprecipitation, or by enxzy- containing 10% fetal calf serum.

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o (7) Incubate 48 hours at 37 C in 5% CO2. Delivery System (Bio-Rad Laboratories GmbH, (8) Examine the cells everyday after the transfer- München, Germany). Biolistic parameters are 15 in. Hg ction. of chamber vacuum, target distance of 3 cm (stage 1), (9) For stable transfection, continue incubate 3 900 psi to 1800 psi particle acceleration pressure, and weeks with changing medium every 2 days. 1.0 µm diameter gold microcarriers (Bio-Rad, USA). Gold microcarriers are prepared, and circular plasmid 3.6 Virus DNA is precipitated onto the gold using methods Viruses are highly adapted to the process of gene recommended by Bio-Rad with the following: 0.6 mg transfer. Viral vectors have the ability to transfer DNA of gold particles carrying ~5 µg of plasmid DNA is to a high fraction of cells, but using virus as the vector used per bombardment. will be potentially arouse cancer leukaemia The detail protocol for the gene gun transfection is (Cavazzana-Calvo, 2004). Common vectors used for described as follows: gene transfer in cell culture are derived from (1) Prepare gold or tungsten particles: 60 mg gold retroviruses. Adenovirus and other agents are used for or tungsten in 1 ml 70% ethanol, centrifuge at 10,000 the gene delivery. rpm for 10 seconds and collect particles, and wash with H2O three times by centrifugation. 3.7 Biolistics (Gene gun, or called microparticle (2) Prepare DNA-coated particles: Mix 50 l bombardment) (about 3 mg) metal, 2.5 l plasmid DNA (about 2.5 Some cells, tissues and intracellular organelles are g), CaCl2 50 l (2.5 M), spermidine 20 l (0.1M). impermeable to foreign DNA, especially plant cells. Vortex and stand for 5 min. Centrifuge, remove biolistics, including particle bombardment, is a supernatant, and add 140 l 70% ethanol over the commonly used method for genetic transformation of pelleted particles, and repeat the ethanol and plants and other organisms. To resolve this problem in centrifugation three times, then add 50 l ethanol. gene transfer, the gene gun was made by Klein at (3) Place a macrocarrier in the metal holder of Cornell University in 1987 (Klein, 1987; Kikkert, gene gun and wash twice with ethanol. 2005). On the gene gun technique, Klein and Sanford (4) Vortex and spread 0.5 mg pellet slurry on the et al published papers, obtained patents and formed a macrocarrier. company called Biolistics (Klein, 1987). (5) Load the macrocarrier into the gene gun, and The gene gun is part of the gene transfer method shoot it. Repeat the shoot until all the areas are shot. . called the biolistic (also known as biobalistic or particle (6) For transient expression, examine cells 48 bombardment) method. In this method, DNA or RNA hours after the shooting, by immunology or other adhere to biological inert particles (such as gold or methods. tungsten). By this method, DNA-particle complex is (7) For stable transfection, continue culture the put on the top location of target tissue in a vacuum transfected cells or tissues. condition and accelerated by powerful shot to the tissue, In our studies, we did gene transfection with the then DNA will be effectively introduce into the target self-design CO2 propelled gene gun (200 psi, distance 3 cells. Uncoated metal particles could also be shot cm with 400 mesh nylon screen) using tungsten particle through a solution containing DNA surrounding the (600 nm diameter) coated with plasmid expressing cell thus picking up the genetic material and anti-ampicillin gene, the plasmid with anti-ampicillin proceeding into the living cells. The efficiency of the gene was transferred into E. Coli cells. gene gun transfer could be depended on the following factors: cell type, cell growth condition, culture 3.8 Laser transfection medium, gene gun ammunition type, gene gun settings As the example of our experiments, UV excimer and the experimental experiences, etc. laser (XeCl2, 308 nm) is used in the gene transfection Briefly for gene gun practice, the target cells or (5 min by a 0.7 0.9, 1.4 or 2.0 mm diameter fiber with tissues on the polycarbonate membranes could be fluence of 45 and 60 mj/mm2 - real laser energy 2.3, positioned in a Biolistic PDS-1000/HE Particle

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5.9, 13.1, 32.0 mj/pulse, 25 Hz) (CVX-300 Excimer Laser System, Spectranetics Corporation, Colorado The current century will bring tremendous Springs, CO, USA). Also, we used to make changes to the science, technology, and the practice of experiments with Nd:Yag, Ho:Yag in the gene medicine (Lushai, 2002). Gene therapy is part of a transfection. All the methods of excimer, Nd:Yag and growing field in molecular medicine, which will gain Ho:Yag laser transfection are effective. importance in the treatment of human diseases (Gunther, 2005). As a critical topic, gene transfection 3.9 Transfection enhanced by elevated tempera- gives people the hope to treat many diseases but it also ture could create dangerous species in the earth, so that it Our studies show that high temperature enhances attracts plenty attention by the whole human society the gene transfection. In our experiments, rat heart (Lanza, 2002). This simply means that the success of muscle cells were cultured in medium 199 with 10% gene transfer technique will be benefit for the FBS and human aorta smooth muscle cells were civilization, and also create the danger for the life in cultured in F12K medium. Human interleukin-2 gene the earth either (Schiemann, 2003). Gene transfection was transfected into rat heart cells and swine growth procedures are used in the critic procedure animal hormone gene was transfected into human aorta clone (Chesne, 2002; Heyman, 2002), and the animal smooth muscle cells by calcium phosphate clone is challenged by the religious groups and ethnic coprecipitation at various temperatures: 23ºC, 37ºC and extremists (Houdebine, 2003). As our personal views, 43ºC. Transfected interleukin-2 and swine growth no matter how big challenges from whatever aspects, hormone expressions were detected using an indirect the gene transfection and animal clone will develop ELISA. The heated cultured rat myocytes had a quickly. The world is a complex place composed by significantly higher expression of the transfected different people. For the science and technology such interleukin-2 gene. Ambient temperature rise to 43oC as gene transfer and animal clone, no country can for up to 30 min provided greater transient transfection prevent other countries from the pursuing. We need to of the interleukin-2 gene when compared to ambient develop the technique even if the technique could be temperatures at 37oC and 23oC (p<0.01). The greatest used in the danger action, and we need to consider the effects occurred within 10 min of incubation and social effects of a technique when we develop it either. persisted up to 30 min. These results suggest that even Science development will be benefit to all the a few degrees of ambient temperature rise can human society. As the gene therapy developing, many significantly increase gene transfer into muscle cells. more desperate diseases could be cured and many This may be of value when using gene therapy with human livings could be saved, such as the life of Pope transfection procedures (Ma, 2004b; Ma, 2004c). John Paul II and Terri Schindler-Schiavo. Hope that the gene transfer techniques described in this article could 3.10 Plant gene transfer be useful for the researches in the gene therapy field Agriculture and plant breeding relied solely on the and help to advance the life science study. accumulated experience of generations of farmers and breeders that is, on sexual transfer of genes between Correspondence to: plant species. However, developments of plant Hongbao Ma molecular biology and genomics now give us access to B410 Clinical Center knowledge and understanding of plant genomes and the Michigan State University possibility of modifying them. There are two most East Lansing, MI 48824, USA powerful technologies for transferring gene into plants: Telephone: (517) 432-0623 Agrobacterium-mediated transformation and biolistics. Email: [email protected] As plants have cell wall, the biolistics is very useful in the plant gene transfer (Rasco-Gaunt, 2001). References [1] Arat S, Rzucidlo SJ, Gibbons J, Miyoshi K, Stice SL. 4 Discussion Production of transgenic bovine embryos by transfer of

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transfected granulosa cells into enucleated oocytes. Mol Reprod [18] Ma H. Technique of Animal Clone. Nature and Science Dev 2001;60(1):20-6. 2004(a);2(1):29-35. [2] Avery OT, MacLeod CM, McCarty M. Studies on the chemical [19] Ma H, Chi C, Abela GS. Increase in ambient temperature nature of the substance inducing transformation of enhances gene transfer into human smooth muscle cells. pneumococcal types. J Exp Med 1944;79:137-58. FASEB Journal 2004(b);18(8):C293. [3] Bordignon V, Keyston R, Lazaris A, Bilodeau AS, Pontes JH, [20] Ma H, Chi C, Abela GS. Increased ambient temperature Arnold D, Fecteau G, Keefer C, Smith LC. Transgene enhances human interleukin-2 gene transfer into cultured expression of green fluorescent protein and germ line myocytes. Journal of Investigative Medicine transmission in cloned calves derived from in vitro-transfected 2004(c);52(2):S390. somatic cells. Biol Reprod 2003;68(6):2013-23. [21] McCutchan JH, Pagano JS. Enchancement of the infectivity of [4] Campbell KH. Nuclear transfer in farm animal species. Semin simian virus 40 deoxyribonucleic acid with diethylaminoethyl- Cell Dev Biol 1999;10(3):245-52. dextran. J Natl Cancer Inst 1968;41(2):351-7. [5] Cavazzana-Calvo M, Thrasher A, Mavilio F. The future of gene [22] Nead MA, McCance DJ. Poly-L-ornithine-mediated therapy. Nature 2004;427(6977):779-81. transfection of human keratinocytes. J Invest Dermatol [6] Chaney WG, Howard DR, Pollard JW, Sallustio S, Stanley P. 1995;105(5):668-71. High-frequency transfection of CHO cells using polybrene. [23] Pagano JS, Vaheri A. Enhancement of infectivity of poliovirus Somat Cell Mol Genet 1986;12(3):237-44. RNA with diethylaminoethyl-dextran (DEAE-D). Arch [7] Chesne P, Adenot PG, Viglietta C, Baratte M, Boulanger L, Gesamte Virusforsch 1965;17(3):456-64. Renard JP. Cloned rabbits produced by nuclear transfer from [24] Rasco-Gaunt S, Riley A, Cannell M, Barcelo P, Lazzeri PA. adult somatic cells. Nat Biotechnol 2002;20(4):366-9. 2001. Procedures allowing the transformation of a range of [8] Griffith F. The significant of pneumoccopal types. Hyg J European elite wheat (Triticum aestivum L.) varieties via 1928;27:113. particle bombardment. J Exp Bot 2001;52(357):865-74. [9] Gunther M, Wagner E, Ogris M. Specific targets in tumor [25] Sambrook J, Russell DW. Molecular cloning: a laboratory tissue for the delivery of therapeutic genes. Curr Med Chem manual. Cold Spring Harbor Laboratory Press, Cold Spring Anti Canc Agents 2005;5(2):157-71. Harbor, NY, USA. 2001;3(16):16.1-16.62. [10] Hawley-Nelson P, Ciccarone V, Gebeyehu G, Jessee J, Felgner [26] Schiemann J. New science for enhanced biosafety. Environ P. FOCUS 1993;15:73. Biosafety Res 2003;2(1):37-41. [11] Hershey AD, Chase D. Independent functions of viral protein [27] Shih PJ, Evans K, Schifferli KP, Ciccarone V, Lichaa F, and nuclei acid in growth of bacteriophage. J Gen Physiol Masoud MJ, Hawley-Nelson P. FOCUS 1997;19:52. 1952;36:39-56. [28] Umemoto Y, Sasaki S, Kojima Y, Kubota H, Kaneko T, [12] Heyman Y, Zhou Q, Lebourhis D, Chavatte-Palmer P, Renard Hayashi Y, Kohri K. Gene transfer to mouse testes by JP, Vignon X. Novel approaches and hurdles to somatic cloning electroporation and its influence on spermatogenesis. J Androl in cattle. Cloning Stem Cells 2002;4(1):47-55. 2005;26(2):264-71. [13] Houdebine LM. Animal transgenesis and Cloning. John Wiley [29] Uzogara SG. The impact of genetic modification of human & Sons, Inc. Hoboken, NJ, USA, 2003:34-73. foods in the 21st century: a review. Biotechnol Adv [14] Lanza RP, Dresser BL, Damiani P. Cloning Noah’s ark, in 2000;18(3):179-206. Understanding Cloning. Scientific American, Inc. and Byron [30] Warden D, Thorne HV. The infectivity of polyoma virus DNA Press Visual Publications, Inc, 2002:24-35. for mouse embryo cells in the presence of diethylaminoethyl- [15] Kikkert JR, Vidal JR, Reisch BI. Stable transformation of plant dextran. J Gen Virol 1968;3(3):371-7. cells by particle bombardment/biolistics. Methods Mol Biol 2005;286:61-78. [16] Klein TM, Wolf ED, Wu R, Sanford JC. Hugh-velocity microprojeectiles for delivering nucleic acids into living cells. Nature 1987’327:70-3. [17] Lorence A, Verpoorte R. Gene transfer and expression in plants. Methods Mol Biol 2004;267:329-50.

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International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 7 Number 07 (2018) Journal homepage: http://www.ijcmas.com

Review Article https://doi.org/10.20546/ijcmas.2018.707.312

Methods of Plant Transformation- A Review

G. Keshavareddy1*, A.R.V. Kumar1 and Vemanna S. Ramu2

1Department of Entomology, 2Department of Crop Physiology, University of Agricultural Sciences, GKVK, Bengaluru-560065, Karnataka, India

*Corresponding author

ABSTRACT

K e yw or ds Plant transformation is now a core research tool in plant biology and a practical tool for transgenic plant development. There are many verified methods for stable introduction of Transformation, novel genes into the nuclear genomes of diverse plant species. As a result, gene transfer Transgenic, Gene, and regeneration of transgenic plants are no longer the factors limiting the development Agrobacterium , and application of practical transformation systems for many plant species. However, the Particl e desire for higher transformation efficiency has stimulated work on not only improving bombardment Article Info various existing methods but also in inventing novel methods. The most published techniques for gene transfer into plant cells were dismissed as either disproven or Accepted: impractical for use in routine production of transgenic plants. In many laboratories, 20 June 2018 virtually all the transformation work relies on particle bombardment with DNA coated Available Online: microprojectiles or Agrobacterium mediated transformation for gene transfer to produce 10 July 2018 transgenic plants from a range of plant species. Introduction Among the various r-DNA technologies, genetically modified plants expressing δ- Plant genetic transformation permits direct endotoxin genes from Bacillus thuringiensis introduction of agronomically useful genes (Bt), protease inhibitors and plant lectins have into important crops and offers a significant been successfully developed, tested and tool in breeding programs by producing novel demonstrated to be highly viable for pest and genetically diverse plant materials. The management in different cropping systems directed desirable gene transfer from one during the last decade and a half (Gatehouse, organism to another and the subsequent stable 2008). Insect resistant crops have been one of integration and expression of a foreign gene in the major successes of applying plant genetic the genome is referred to as ‘Genetic engineering technology to agriculture. Most Transformation’. The transferred gene is of the plant derived genes produce chronic known as ‘transgene’ and the organisms that rather than toxic effects and many insect pests are developed after a successful gene transfer are less or not sensitive to most of these are known as ‘transgenics’ (Babaoglu et al., factors. Therefore, the genes for δ-endotoxins 2000). are expected to provide better solutions.

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Advances in biotechnology have provided 2002. Success achieved in cotton has served several unique opportunities that include as an excellent model to emulate in many access to various plant transformation other crops such as rice, wheat, pulses and techniques, novel and effective molecules, oilseeds that have the potential to make ability to change the levels of gene expression, agriculture a viable profession for the peasants capability to change the expression pattern of of India. genes, and develop transgenics with different insecticidal genes. With the advent of genetic Transformation studies transformation techniques based on recombinant DNA technology, it is now Plant transformation is now a core research possible to insert foreign genes that confer tool in plant biology and a practical tool for resistance to insects into the plant genome transgenic plant development. There are (Bennett, 1994). To sustain the crop yield many verified methods for stable introduction potential and to meet the growing demand for of novel genes into the nuclear genomes of food, crop productivity needs to be increased. diverse plant species. The capacity to However, in most crops it is believed that the introduce and express diverse foreign genes in genetic potential has been fully exploited for plants, first described for tobacco in 1984 yield increase. As a result, any improvement (DeBlock et al., 1984; Horsch et al., 1984; in productivity has to revolve around the Paszkowski, 1984) has been extended to many reduction of losses due to pests and diseases plant species in at least 35 families. under optimal nutrition and abiotic factors. Recombinant DNA technology coupled with Gene transfer successes include most major plant tissue culture has helped develop novel economic crops, vegetables and medicinal options for the economical management of plants. As a result, gene transfer and various kinds of biotic stresses including regeneration of transgenic plants are no longer insect pests. These technologies would be of the factors limiting the development and immense value in reducing the losses caused application of practical transformation systems by biotic stresses, including insect pests. for many plant species. The techniques have continued to evolve to over come a great Transgenic plants display considerable variety of barriers experienced in the early potential to benefit both developed and phases of the development in the field of plant developing countries. Transgenic plants transformation. expressing insecticidal Bt proteins alone or in conjunction with proteins providing tolerance Transformation methods to herbicide are revolutionizing agriculture (Shelton et al., 2002). The use of such crops Gene delivery systems involve the use of with input traits for pest management, several techniques for transfer of isolated primarily insects and herbicide resistance, has genetic materials into a viable host cell. At risen dramatically since their first introduction present, there are two classes of delivery in the mid 1990s. systems (Table 1): (a) Non-biological systems (which include chemical and physical India, the largest cotton growing country in methods) and (b) Biological systems. The the world has increased productivity by up to desire for higher transformation efficiency has 50% while reducing the insecticide sprays by stimulated work on not only improving half, with environmental and health various existing methods but also in inventing implications, besides increased income to novel methods. cultivators after introduction of Bt cotton in 2657

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Biological requirements for transformation (6) Simple integration patterns and low copy number of introduced genes, to minimize the The essential requirements in a gene transfer probability of undesired gene disruption at system for production of transgenic plants are: insertion sites, or multicopy associated transgene silencing. Availability of a target tissue including cells competent for plant regeneration. (7) Stable expression of introduced genes in the pattern expected from the chosen gene A method to introduce DNA into those control sequences (DeBlock, 1993). regenerable cells and When tested against the above criteria, most A procedure to select and regenerate published techniques for gene transfer into transformed plants at a satisfactory frequency. plant cells must be dismissed as either disproven or impractical for use in routine Practical requirements for transformation production of transgenic plants. As a result, in many laboratories, virtually all the Beyond the biological requirements to achieve transformation work relies on Particle transformation and the technical requirements bombardment with DNA coated for verification of reproducible microprojectiles or Agrobacterium mediated transformation, desired characteristics to be transformation for gene transfer to produce considered in evaluating alternative techniques transgenic plants in a range of plant species or developing new ones for cultivar (Birch, 1997). improvement include: Non-biological based transformation (1) High efficiency, economy, and reproducibility, to readily produce many Particle bombardment/Biolistics independent transformants for testing. Particle bombardment was first described as a (2) Safety to operators, avoiding procedures, method for the production of transgenic plants or substances requiring cumbersome in 1987 (Sanford et al., 1987) as an alternative precautions to avoid a high hazard to operators to protoplast transformation and especially for (e.g. potential carcinogenicity of Silicone transformation of more recalcitrant cereals. carbide whiskers). Unique advantages of this methodology compared to alternative propulsion (3) Technical simplicity, involving a minimum technologies are discussed elsewhere in terms of demanding or inherently variable of range of species and genotypes that have manipulations, such as protoplast production been engineered and the high transformation and regeneration. frequencies for major agronomic crops (McCabe and Christou, 1993). (4) Minimum time in tissue culture, to reduce associated costs and avoid undesirable In plant research, the major applications of somaclonal variation. biolistics include transient gene expression studies, production of transgenic plants and (5) Stable, uniform (nonchimeric) inoculation of plants with viral pathogens transformants for vegetatively propagated (Southgate et al., 1995; Sanford, 2000; Taylor species, or fertile germline transformants for and Fauquet, 2002). sexually propagated species. 2658

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Gene constructs for biolistics can be in the revolutionary. Genetic transformation of form of circular or linear plasmids or a linear plants was viewed as a prospect. In retrospect, expression cassette. Embryogenic cell Agrobacterium was the logical and natural cultures are likely the best explants to use for transformation candidate to consider since it biolistic transformation because they can be naturally transfers DNA (T-DNA) located on spread out as uniform targets of cells and have the tumor inducing (Ti) plasmid into the high recovery capacity (Kikkert et al., 2004). nucleus of plant cells and stably incorporates Rice transformation has also been successfully the DNA into the plant genome (Chilton et al., achieved via the bombardment of 1977). Now forty five years later, this method embryogenic calli (Li et al., 1993; Sivamani et has been the most widely used and powerful al., 1996; Cao et al., 1992; Zhang et al., technique for the production of transgenic 1996), in which transformation efficiency has plants. However, there still remain many been raised to 50% (Li et al., 1993). Particle challenges for genotype independent bombardment has emerged as a reproducible transformation of many economically method for wheat transformation (DeBlock et important crop species, as well as forest al., 1997; Bliffeld et al., 1999) and the first species (Stanton, 2003; De la Riva et al., stable transformation in a commercially 1998). important conifer species (Picea glauca) was achieved via embryogenic callus tissue as Despite the development of other non- explant (Ellis et al., 1993). biological methods of plant transformation (Shillioto et al., 1985; Uchimiya et al., 1986; However, particle bombardment has some Sanford, 1988; Arenchibia et al., 1992, 1995), disadvantages. The transformation efficiency Agrobacterium mediated transformation might be lower than with Agrobacterium remains popular and is among the most mediated transformation and it is more costly, effective. This is especially true among most as well. Intracellular targets are random and dicotyledonous plants, where Agrobacterium DNA is not protected from damage. As a is naturally infectious. Agrobacterium result, many researchers have avoided particle mediated gene transfer into monocotyledonous bombardment method because of the high plants was thought to be not possible. frequency of complex integration patterns and However, reproducible and efficient multiple copy insertions that could cause gene methodologies have been established for rice silencing and variation of transgene (Hiei et al., 1994), banana (May et al., 1995, expression (Dai et al., 2001; Darbani et al., corn (Ishida et al., 1996), wheat (Cheng et al., 2008). 1997), sugarcane (Arencibia et al., 1998), forage grasses such as Italian ryegrass (Lolium Biological gene transfer multiflorum) and tall fescue (Festuca arundinacea) (Bettany et al., 2003). Among Agrobacterium mediated transformation the commercially important conifers, hybrid larch was the first to be stably transformed via The natural ability of the soil bacteria, co-cultivation of embryogenic tissue with A. Agrobacterium tumefaciens and tumefaciens (Levee et al., 1997). Agrobacterium rhizogenus, to transform host Subsequently, this method was successfully plants has been exploited in the development applied to several species of spruce of transgenic plants. In 1970s the prospect of (Klimaszewska et al., 2001; Charity et al., using A. tumefaciens for the rational gene 2005; Grant et al., 2004). transfer of exogenous DNA into crops was

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Methods relative to transformation targets can So why have all these other methods emerged be classified into two categories: (a) those in the past 20-30 years, if we already have requiring tissue culture and (b) in planta efficient transformation techniques in methods. Agrobacterium and biolistics? There are two reasons. First of all, there is hope that a more In tissue culture systems for plant efficient and less expensive method would be transformation, the most important developed. The second and most important requirement is a large number of regenerable reason is the biolistics and Agrobacterium are cells that are accessible to the gene transfer patented. treatment and that will retain the capacity for regeneration for the duration of the necessary In planta transformation target preparation, cell proliferation and selection treatments. A high multiplication Although successful plant regeneration ratio from a micropropagation system does not methods have been developed, the technology necessarily indicate a large number of has not provided regeneration in several other regenerable cells accessible to gene transfer crops for use in transformation protocols (Livingstone and Birch, 1995). Some time which is a serious limitation to the gene transfer into potentially regenerable cells exploitation of gene transfer technology to its may not allow recovery of transgenic plants if full potential. In the light of this major the capacity for efficient regeneration is short constraint, it becomes necessary to evolve lived (Ross et al., 1995). Further, tissue transformation strategies that do not depend culture based methods can lead to unwanted on tissue culture regeneration or those that somaclonal variations such as alterations in substantially eliminate the intervening tissue cytosine methylation, induction of point culture steps. In planta transformation mutations and various chromosomal methods provide such an opportunity. aberrations (Phillips et al., 1994; Singh, 2003; Methods that involve delivery of transgenes in Clough, 2004). On the other hand, realization the form of naked DNA directly into the intact of whole plant transformants has been a plants are called as in planta transformation problem in a large number of crop species as methods. These methods exclude tissue these plants have proven to be highly culture steps, rely on simple protocols and recalcitrant in vitro. As a result, other required short time in order to obtain entire strategies are being evolved wherein the tissue transformed individuals. culture component is obviated in the procedure and these are known as in planta In many cases in planta methods have targeted methods. meristems or other tissues with the assumption that at fertilization, the egg cell accepts the Plant genetic transformation is of particular donation of an entire genome from the sperm benefit to molecular genetic studies, crop cell that will ultimately give rise to zygotes improvement and production of (Chee and Slighton, 1995; Birch, 1997) and pharmaceutical materials. Agrobacterium- therefore is the right stage to integrate based methods are usually superior for many transgenes. For non-tissue culture based species including dicots and monocots. The approaches of in planta transformation, others are typically not done on a routine basis Agrobacterium co-cultivation or (Table 2). Biolistics is by far the most widely microprojectile bombardment have been used direct transformation procedure both directed to transform cells in or around the experimentally in research and commercially. apical meristems (Chee and Slighton, 1995;

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Birch, 1997). Injection of naked DNA into Bent, 1998), transformation of germinating ovaries has also been reported to produce seeds (Feldmann and Marks, 1987) and floral transformed progeny (Zhou et al., 1983). dipping (Clough and Bent, 1998). Other plants that were successfully subjected by vacuum Arabidopsis thaliana was the first plant that infiltration include rapeseed, Brassica saw successful in planta transformation. campestris and radish, Raphanus sativus (Ian Early stages of success in Arabidopsis and Hong, 2001; Desfeux et al., 2000). The transformation came from the work of labor intensive vacuum infiltration process Feldmann and Marks (1987). Transformation was eliminated in favor of simple dipping of rates greatly improved when Bechtold et al. developing floral tissues (Clough and Bent, (1993) inoculated plants that were at the 1998). Also, the results indicate that the floral flowering stage. At present, there are very spray method of Agrobacterium can achieve few species that can be routinely transformed high rates of in planta transformation in the absence of a tissue culture based comparable to the vacuum infiltration and regeneration system. Arabidopsis can be floral dip methods (Chung et al., 2000). transformed by several in planta methods including vacuum infiltration (Clough and

Table.1 DNA delivery methods available to produce plant transformants

Plant transformation Non-biological based transformation Biological gene transfer (Direct method) (Indirect method) A) DNA transfer in protoplasts 1) Chemically stimulated DNA uptake 1) Agrobacterium mediated by protoplast transformation 2) Electroporation 3) Lipofection Primarily two methods 4) Microinjection 5) Sonication a) Co-cultivation with the explants tissue B) DNA transfer in plant tissues 1) Particle bombardment / Biolistics b) In planta transformation 2) Silicon carbide fiber mediated gene transfer 2) Transformation mediated by viral 3) 3) Laser microbeam (UV) induced vector genetransfer (Birch et al., 1997)

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Table.2 Summary of gene delivery methods and their features

Gene delivery Transformation Range of Tissue Type of Remarks method efficiency transformable plant culture phase explant species Electroporation Low to high Unrestricted With and Protoplasts, Fast, simple and inexpensive in without tissue meristems or contrast with biolistics culture phase pollen grains

Lipofection Low Recoverable species With tissue Protoplast High efficiency with combination of from protoplast culture phase PEG based method, simple and non- toxic

Microinjection High Recoverable species With tissue Protoplast Very slow, precise, single cell from protoplast culture phase targeting possibility, requires high skill, the chimeric nature of transgenic plants and ability of whole chromosome transformation

Sonication Low Unrestricted With and Protoplast cells, Effective to transfect by virus particles without tissue tissues and and able to increase the culture seedlings Agrobacterium based transformation efficiency

Efficient for viral infection, complex Particlebombar High Unrestricted With and Intact tissue or integration patterns, without dment without tissue microspores specialized vectors and backbone free culture phase integration

(Darbani et al., 2008)

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Gene delivery Transformation Range of Tissue culture Type of explant Remarks method efficiency transformable plant phase species Silicon carbide Low to high Unrestricted With tissue Variety of cell Rapid, inexpensive and easy to set up mediate culture types transformation

Laser beam Low Unrestricted With tissue Variety of cell Rapid and simple mediated culture phase types transformation

Agrobacterium High and stable Many species, With and Different intact Possibility of Agroinfection, mediated specially without tissue cells, tissues or combination with sonication and method dicotyledonous culture whole plant biolistic methods and transgene size plants method up to 150 kb

Virus based High and Virus host specific With tissue In planta Rapid, inducible expression and with method transient limitation culture inoculation mosaic status

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Utilizing naked DNA, cotton transformants a practical possibility for experimenting and were recovered following injection of DNA producing viable transformants. However, into the axil placenta about a day after self- the optimization of Agrobacterium-plant pollination (Zhou et al., 1983). Similarly, a interaction is crucial for efficient mixture of DNA and pollen was either applied transformation. Many factors including type to receptive stigmatic surfaces or DNA was of explant are important and they must be injected directly into rice floral tillers, or suitable to allow the recovery of whole soybean seeds were imbibed with DNA transgenic plants (De la Ravi et al., 1998; (Trick and Finer, 1997; Langridge, 1992). Opabode 2006; Cheng, et al., 1997; Jones et These procedures, intriguing as they are, are al., 2005; Darbani et al., 2008). impractical at present because of their low reproducibility. Although, biotechnological advances, have provided many technologies for gene transfer Recent studies with Agrobacterium into plant cells, virtually all the inoculation of germinating seeds of rice has transformation work rely only on particle provided transformation efficiencies higher bombardment with DNA coated than 40% (Supartana et al., 2005), while microprojectiles or Agrobacterium mediated providing 4.7 to 76% efficiency for the flower transformation for gene transfer to produce infiltration method and from 2.9 to 27.6% transgenic plants. The review thus efficiency for the seedling infiltration method overwhelmingly emphasizes the importance (Trieu et al., 2000). of this method.

Crop species that were successfully References transformed by injuring the apical meristem of the differentiated embryo of the Arencibia, A., P. Molina, C. Gutierrez, A. germinating seeds and then infecting with Fuentes, V. Greenidge, E. Menendez, Agrobacterium include peanut, Arachis G. De la Riva and Housein, G. S. hypogaea L. (Rohini and Rao, 2000b & 1992. Regeneration of transgenic 2001), sunflower, Helianthus annuus L. (Rao sugarcane (Saccharum officinarum L.) and Rohini, 1999), safflower, Carthamus plants from intact meristematic tissue tinctorius L. (Rohini and Rao, 2000a), field transformed by electroporation. bean, Dolichos lablab L. (Pavani, 2006), and Biotechnologia Aplicada 9: 156-165. cotton, Gossypium sp. (Keshamma et al., Arencibia, A. D., E. R. Carmona, P. Tellez, 2008). Maize, Zea mays L., was transformed M. T. Chan, S. M. Yu, L. E. Trujillo by treating the silks with Agrobacterium and and Oramas, P. 1998. An efficient afterwards pollinated with the pollen of the protocol for sugarcane (Saccharum sp. same cultivar (Chumakov et al., 2006). L.) transformation mediated by Agrobacterium tumefaciens. The above successes have in fact provided a Transgenic Res. 7(3): 213-222. great leverage for easy development of Babaoglu, M., M. R. Davey and Power, J. B. transgenic pants, as the methodology is 2000. Genetic engineering of simple, cost effective, does not call for high grainlegumes: key transformation infrastructural requirement even to handle events. Agri. Biotech. Net. 2:1–12. recalcitrant crops such as groundnut. Thus Bechtold, N., J. Ellis and Pelletier, G. 1993. the technology of gene transfer for the In planta Agrobacterium mediated development of recalcitrant crops has become gene transfer by infiltration of adult

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Arabidopsis thaliana plants. C. R. Cheng, M., J. E. Fry, S. Z. Pang, H. P. Zhou, Acad. Sci. (Paris) Life Sci. 316: 1194- C. M. Hironaka, D. R. Duncan, W. 1199. Conner and Wan, Y. C. 1997. Genetic Bennett, J. 1994. DNA-based techniques for transformation of wheat mediated by control of rice insects and diseases: Agrobacterium tumefaciens. Plant Transformation, gene tagging and Physiol. 115(3): 971-980. DNA fingerprinting. In: Rice pest Chilton, M. D., M. H. Drummond, D. J. science and management, P.S. Teng, Merio, D. Sciaky, A. L. Montoya, M. K.L. Heong and K. Moody (eds.), P. Gordon and Nester, E. W. 1977. International Rice Research Institute, Stable incorporation of plasmid DNA Los Banos, Philippines, pp.147-172. into higher plant cells: The molecular Bettany, A. J. E., S. J. Dalton, E. J. Timms, basis of crown gall tumorigenesis. B. Manderyck, M. S. Dhanoa And Cell 11(2): 263-271. Morris, P. 2003. Agrobacterium Chung, M. H., M. K. Chen and Pan, S. M. tumefaciens mediated transformation 2000. Floral spray transformation can of Festuca arundinacea (Sahreb.) and efficiently generate Arabidopsis. Lolium multiflorum (Lam.). Plant Cell Transgenic Res. 9(6): 471-486. Rep. 21(5): 437-444. Clough, S. J. and Bent, A. F. 1998. Floral dip: Birch, R. G. 1997. Plant transformation: a simplified method for problems and strategies for practical Agrobacterium mediated application. Ann. Rev. Plant Physiol. transformation of Arabidopsis Plant Mol. Biol. 48: 297-326. thaliana. Plant J. 16(6): 735-743. Bliffeld, M., J. Mundy, I. Potrykus and Clough, S. J. 2004. Floral Dip: Futterer, J. 1999. Genetic engineering Agrobacterium-mediated germ line of wheat for increased resistance to transformation. In: Methods in powdery mildew disease. Theor. molecular biology. Transgenic plants: Applied Genet. 98(7): 1079-1086. methods and protocols, Pena, L. (Ed.). Cao, J., X. Duan, D. McElroy and Wu, R. Vol. 286. Humana Press Inc: Totowa, 1992. Regeneration of herbicide NJ. Pp: 91-102. resistant transgenic rice plants Dai, S., P. Zheng, P. Marmey, S. Zhang, W. following microprojectile mediated Tian, S. Chen, R. N. Beachy and transformation of suspension culture Fauquet, C. 2001. Comparative cells. Plant Cell Rep. 11(11): 586-591. analysis of transgenic rice plants Charity, J. A., L. Holland, L. Grace and obtained by Agrobacterium mediated Walter, C. 2005. Consistent and stable transformation and particle expression of the nptII, uidA and bar bombardment. Mol. Breed. 7(1): 25- genes in transgenic Pinus radiate after 33. Agrobacterium tumefaciens mediated Darbani, B., S. Farajnia, M. Toorchi, S. transformation using nurse cultures. Zakerbostanabad, S. Noeparvar and Plant Cell Rep. 23(9): 606-619. Stewart, C. N. Jr. 2008. DNA-delivery Chee, P. P. and Slighton, J. L. 1995. methods to produce transgenic plants. Transformation of soybean (Glycine Biotechnology 26: 1-18. max) via Agrobacterium tumefaciens DeBlock, M. 1993. The cell biology of plant and analysis of transformed plants. In: transformation: current state, Agrobacterium protocols: Methods problems, prospects and the Mol. Biol. 44: 101-109. implications for plant breeding.

2665

Int.J.Curr.Microbiol.App.Sci (2018) 7(7): 2656-2668

Euphytica 71:1–14. Hiei, Y., S. Ohta, T. Komari and Kumashiro, DeBlock, M., H. L. Estrella, M. van Montagu, T. 1994. Efficient transformation of J. Schell and Zambryski, P. 1984. rice (Oryza sativa) mediated by Expression of foreign genes in Agrobacterium and sequence analysis regenerated plants and their progeny. of the boundaries of the T-DNA. Plant EMBO J. 3:1681–1689. J. 6(2): 271-282. DeBlock, M., D. Debrouwer and Moens, T. Horsch, R. B., R. T. Fraley, S. G. Rogers, P. 1997. The development of a nuclear R. Sanders and Lloyd, A. 1984. male sterility system in wheat. Inheritance of functional foreign genes Expression of the barnase gene under in plants. Science 223:496–498. the control of tapetum specific Ian, S. C. and Hong, G. N. 2001. Transgenic promoters. Theor. Applied Genet. radish (Raphanus sativus L. 95(1-2): 125-131. longipinnatus Bailey) by floral-dip De la Riva, G. A., J. G. Cabrera, R. V. Padron method plant development and and Pardo, C. A. 1998. Agrobacterium surfactant are important in optimizing tumefaciens: A natural tool for plant transformation efficiency. Transgenic transformation. Elect. J. Biotechnol. Res. 10(4): 363-371. 1(3): 118-133. Ishida, Y., H. Saito, S. Ohta, Y. Hiei, T. Desfeux, C., S. J. Clough and Bent, A. F. Komari and Kumashiro, T. 1996. 2000. Female reproductive tissues are High efficiency transformation of the primary target of Agrobacterium- maize (Zea mays L.) mediated by mediated transformation by the Agrobacterium tumefaciens. Nat. Arabidopsis floral –dip method. Plant Biotechnol. 4: 745-750. Physiol. 123(3): 895-904. Jones, H. D., A. Doherty and Wu, H. 2005. Ellis, D. D., D. E. McCabe, S. McInnis, R. Review of methodologies and protocol Ramachandran, D. R. Russell, K. for the Agrobacterium-mediated Wallace, B. J. Martinell, D. R. transformation of wheat. Plant Roberts, K. F. Raffa and McCown, B. Methods 1: 5. H. 1993. Stable transformation of Keshamma, E., S. Rohini, K. S. Rao, B. Picea glauca by particle acceleration. Madhusudhan and Udayakumar, M. BioTechnology 11(1): 84-89. 2008. Tissue culture independent in Feldmann, K. A. and Marks, M. D. 1987. planta transformation strategy: an Agrobacterium mediated Agrobacterium tumefaciens-mediated transformation of germinating seeds of gene transfer method to overcome Arabidopsis thaliana : A non-tissue recalcitrance in cotton (Gossypium culture approach. Mol. Gen. Genet. hirsutum L.). J. Cotton Sci. 12: 264- 208: 1-9. 272. Gatehouse, J. A. 2008. Biotechnological Kikkert, J. R., J. R. Vidal and Reisch, B. I. prospects for engineering insect- 2004. Stable transformation of plant resistant plants. Plant Physiol. 146: cells by particle bombardment/ 881-887. Biolistics. In: Methods in molecular Grant, J. E., P. A. Cooper and Dale, T. M. biology. Transgenic plants: Methods 2004. Transgenic Pinus radiata from and protocols, Pena, L. (Ed.). Vol. Agrobacterium tumefaciens –mediated 286, Humana Press Inc: Totowa, NJ., transformation of cotyledons. Plant pp:61-78. Cell Rep. 22(12): 894-899. Klimaszewska, K., D. Lachance, G. Pelletier,

2666

Int.J.Curr.Microbiol.App.Sci (2018) 7(7): 2656-2668

M. A. Lelu and Seguin, A. 2001. Pavani, C. 2006. Development and Regeneration of transgenic Picea characterisation of transgenics over glauca, P. mariana and P. abies after expressing cry genes in field bean co-cultivation of embryogenic tissue against Helicoverpa armigera with Agrobacterium tumefaciens. In (Hubner). M. Sc. Thesis, University of Vitro Cell Dev. Biol. Plant 37(6): 748- Agricultural Sciences, Bangalore, 755. India pp.99. Langridge, P. 1992. Transformation of cereals Paszkowski, J., R.D. Shillito, M. Saul, V. via Agrobacterium and the pollen Mandak, T. Hohn, B. Hohn and pathway: a critical assessment. Plant Potrykus, I. 1984. Direct gene transfer J. 2: 631-638. to plants. EMBO J. 3: 2717-2722. Levee, V., M. A. Lelu, L. Jouanin, D. Comu Phillips, R. L., S. M. Kaeppler and Olhoft, P. and Pilate, G. 1997. Agrobacterium 1994. Genetic instability of plant tumefaciens mediated transformation tissue cultures: Breakdown of normal of hybrid larch (Larix kaempferi L. controls. Proc. Natl. Acad. Sci. deciduas) and transgenic plant 91(12): 5222-5226. regeneration. Plant Cell Rep. 16(10): Rao, K. S. and Rohini, V. K. 1999. 680-685. Agrobacterium-mediated Li, L., R. Qu, A. Kochko de, C. M. Fauquet transformation of sunflower and Beachy, R. N. 1993. An improved (Helianthus annus L.): A simple rice transformation system using the protocol. Ann. Bot. 83: 347-354. biolistic approach. Plant Cell Rep. Rohini, V. K. and Rao, K. S. 2000a. Embryo 12(1): 50-55. transformation, a practical approach Livingstone, D. M. and Birch, R. G. 1995. for realizing transgenic plants of Plant regeneration and safflower (Carthamus tinctorius L.). microprojectile-mediated gene transfer Ann. Bot. 86: 1043-1049. in embryonic leaflets of peanut Rohini, V. K. and Rao, K. S. 2000b. (Arachis hypogaea L.). Aust. J. Plant Transformation of peanut (Arachis Physiol. 22:585–591. hypogaea L.): a non–tissue culture May, G. D., R. Afza, H. S. Mason, A. based approach for generating Wiecko, F. J. Novak and Amtzen, C. transgenic plants. Plant Sci. 150: 41- J. 1995. Generations of transgenic 49. banana (Musa acuminata) plants via Rohini, V. K. and Rao, K. S. 2001. Agrobacterium mediated Transformation of peanut (Arachis transformation. Biotechnology 13(5): hypogaea L.) with tobacco chitinase 486-492. gene: variable response of McCabe, D. and Christou, P. 1993. Direct transformants to leaf spot disease. DNA transfer using electric discharge Plant Sci. 160: 889-898. particle acceleration (ACCELLTM Ross, A. H., J. M. Manners and Birch, R. G. technology). Plant Cell Tissue Organ 1995. Embryogenic callus production, Culture 33: 227-236. plant regeneration and transient gene Opabode, J. T. 2006. Agrobacterium- expression following particle mediated transformation of plants: bombardment, in the pasture grass Emerging factors that influence Cenchrus ciliarus (Gramineae). Aust. efficiency. Biotechnol. Mol. Biol. Rev. J. Bot. 43:193–199. 1(1): 12-20. Sanford, J. C., T. M. Klein, E. D. Wolf and

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Int.J.Curr.Microbiol.App.Sci (2018) 7(7): 2656-2668

Allen, N. 1987. Delivery of substances Nogawa, M. Nozue and Kojima, M. into cells and tissues using a particle 2005. Development of simple and bombardment process. Particulate Sci. efficient in planta transformation Technol. 5(1): 27-37. method for rice (Oryza sativa L.) Sanford, J. C. 2000. The development of the using Agrobacterium tumefaciens. J. biolistic process. In vitro Cell Dev. Biosci. Bioengg. 100 (4): 391-397. Biol. Plant 36(5): 303-308. Taylor, N. J. and Fauquet, C. M. 2002. Shelton, A. M., J. Z. Zhao and Roush, R. T. Microparticle bombardment as a tool 2002. Economic, ecological, food in plant science and agricultural safety and Social consequences of the biotechnology. DNA Cell Biol. 21(12): deployment of Bt transgenic plants. 963-977. Annu. Rev. Entomol. 47: 845-881. Trick, H. N. and Finer, J. J. 1997. SAAT: Singh, R. J. 2003. Chromosomal aberrations Sonication-assisted Agrobacterium- in cell and tissue culture. In: Plant mediated transformation. Transgenic cytogenetics, Singh, R. J. (Ed.). CRC Res. 6(5): 329-336. Press: Boca Raton, Fl., pp: 307-326. Trieu, A. T., S. H. Burleigh, I. V. Kardailsky, Sivamani, E., P. Shen, N. Opalka, R. N. I. E. M. Mendoza, W. K. Versaw, L. Beachy and Fauquet, C. M. 1996. A. Blaylock, H. Shin, T. J. Chiou, H. Selection of large quantities of Katagi, G. R. Dewbre, D. Weigel and embryogenic subcultured calli from Harrison, M. J. 2000. Transformation indica rice seeds for production of of Medicago truncatula via infiltration fertile transgenic plants using the of seedlings of flowering plants with biolistic method. Plant Cell Rep. Agrobacterium. Plant J. 22(6): 531- 15(5): 322-327. 541. Southgate, E. M., M. R. Davey, J. B. Power Zhang, S., L. Chen, R. Qu, P. Marmey, R. N. and Marchant, R. 1995. Factors Beachy and Fauquet, C. M. 1996. affecting the genetic engineering of Regeneration of fertile transgenic plants by Microprojectile indica (group 1) rice plants following bombardment. Biotechnol. Adv. 13(4): microprojectile transformation of 631-651. embryogenic suspension culture cell. Stanton, B. G. 2003. Agrobacterium mediated Plant Cell Rep. 15(7): 465-469. plant transformation: The biology Zhou, G. Y., J. Weng, Y. Zeng, J. Huang, S. behind the gene jockeying tool. Qian and Liu, G. 1983. Introduction of Microbiol. Mol. Biol. Rev. 67(1): 16- exogenous DNA into cotton embryos. 37. Meth. Enzymol. 101: 433-481. Supartana, P., T. Shimizu, H. Shioiri, M.

How to cite this article:

Keshavareddy, G., A.R.V. Kumar and Vemanna S. Ramu. 2018. Methods of Plant Transformation- A Review Int.J.Curr.Microbiol.App.Sci. 7(07): 2656-2668. doi: https://doi.org/10.20546/ijcmas.2018.707.312

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MODULE 5- LECTURE 3

GENE TRANSFER TECHNIQUES: PHYSICAL OR MECHANICAL METHODS

5-3.1. Gene transfer techniques

It has been discussed earlier that due to amphipathic nature of the phospholipid bilayer of the plasma membrane, polar molecules such as DNA and protein are unable to freely pass through the membrane. Various physical or mechanical methods are employed to overcome this and aid in gene transfer as listed below-

1. Electroporation 2. Microinjection 3. Particle Bombardment 4. Sonoporation 5. Laser induced 6. Bead transfection

5-3.1.1. Electroporation

• Electroporation is a mechanical method used for the introduction of polar molecules into a host cell through the cell membrane. • This method was first demonstrated by Wong and Neumann in 1982 to study gene transfer in mouse cells. • It is now a widely used method for the introduction of transgene either stably or transiently into bacterial, fungal, plant and animal cells. • It involves use of a large electric pulse that temporarily disturbs the phospholipid bilayer, allowing the passage of molecules such as DNA.

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The basis of electroporation is the relatively weak hydrophobic/hydrophilic interaction of the phospholipids bilayer and ability to spontaneously reassemble after disturbance. A quick voltage shock may cause the temporary disruption of areas of the membrane and allow the passage of polar molecules. The membrane reseals leaving the cell intact soon afterwards.

5-3.1.1(a). Procedure

The host cells and the DNA molecules to be transported into the cells are suspended in a solution. The basic process inside an electroporation apparatus is represented in a schematic diagram (Figure 5-3.1.1(a).).

Figure 5-3.1.1(a). The basic circuit setup of the electroporation apparatus. (Adapted from http://o]pbs.okstate.edu/~melcher/MG/MGW4/MG431.html) • When the first switch is closed, the capacitor charges up and stores a high voltage which gets discharged on closing the second switch. • Typically, 10,000-100,000 V/cm in a pulse lasting a few microseconds to a millisecond is essential for electroporation which varies with the cell size. • This electric pulse disrupts the phospholipid bilayer of the membrane causing the formation of temporary aqueous pores. • When the electric potential across the cell membrane is increased by about 0.5-1.0 V, the charged molecules e.g. DNA migrate across the membrane through the pores in a similar manner to electrophoresis. • The initiation of electroporation generally occurs when the transmembrane voltage reaches at 0.5-1.5 V. The cell membrane discharges with the subsequent

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flow of the charged ions and molecules and the pores of the membrane quickly close reassembling the phospholipid bilayer.

5-3.1.1(b). Applications

Electroporation is widely used in many areas of molecular biology and in medical field. Some applications of electroporation include:

• DNA transfection or transformation Electroporation is mainly used in DNA transfection/transformation which involves introduction of foreign DNA into the host cell (animal, bacterial or plant cell). • Direct transfer of plasmids between cells It involves the incubation of bacterial cells containing a plasmid with another strain lacking plasmids but containing some other desirable features. The voltage of electroporation creates pores, allowing the transfer of plasmids from one cell to another. This type of transfer may also be performed between species. As a result, a large number of plasmids may be grown in rapidly dividing bacterial colonies and transferred to yeast cells by electroporation. • Gene transfer to a wide range of tissues

Electroporation can be performed in vivo for more efficient gene transfer in a wide range of tissues like skin, muscle, lung, kidney, liver, artery, brain, cornea etc. It avoids the vector-specific immune-responses that are achieved with recombinant viral vectors and thus are promising in clinical applications.

5-3.1.1(c). Advantages

• It is highly versatile and effective for nearly all cell types and species. • It is highly efficient method as majority of cells take in the target DNA molecule. • It can be performed at a small scale and only a small amount of DNA is required as compared to other methods.

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5-3.1.1(d). Disadvantages

• Cell damage is one of the limitations of this method caused by irregular intensity pulses resulting in too large pores which fail to close after membrane discharge. • Another limitation is the non-specific transport which may result in an ion imbalance causing improper cell function and cell death.

5-3.1.2. Microinjection

• DNA microinjection was first proposed by Dr. Marshall A. Barber in the early of nineteenth century. • This method is widely used for gene transfection in mammals. • It involves delivery of foreign DNA into a living cell (e.g. a cell, egg, oocyte, embryos of animals) through a fine glass micropipette. The introduced DNA may lead to the over or under expression of certain genes. • It is used to identify the characteristic function of dominant genes.

5-3.1.2(a). Procedure

• The delivery of foreign DNA is done under a powerful microscope using a glass micropipette tip of 0.5 mm diameter. • Cells to be microinjected are placed in a container. A holding pipette is placed in the field of view of the microscope that sucks and holds a target cell at the tip. The tip of micropipette is injected through the membrane of the cell to deliver the contents of the needle into the cytoplasm and then the empty needle is taken out.

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Figure 5-3.1.2(a). Delivery of DNA into a cell through microinjection.

(Adapted from http://www.eplantscience.com/index_files/biotechnology/Genes%20&%20Genetic%20Engineering/Techniques%20of%20Genetic %20Engineering/biotech_microinjection.php)

5-3.1.2(b). Advantages

• No requirement of a marker gene. • Introduction of the target gene directly into a single cell. • Easy identification of transformed cells upon injection of dye along with the DNA. • No requirement of selection of the transformed cells using antibiotic resistance or herbicide resistance markers. • It can be used for creating transgenic organisms, particularly mammals.

5-3.1.3. Particle bombardment

• Prof Sanford and colleagues at Cornell University (USA) developed the original bombardment concept in 1987 and coined the term “biolistics” (short for “biological ballistics”) for both the process and the device. • Also termed as particle bombardment, particle gun, micro projectile bombardment and particle acceleration. • It employs high-velocity micro projectiles to deliver substances into cells and tissues.

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5-3.1.3(a). Uses

• This method is commonly employed for genetic transformation of plants and many organisms. • This method is applicable for the plants having less regeneration capacity and those which fail to show sufficient response to Agrobacterium- mediated gene transfer in rice, corn, wheat, chickpea, sorghum and pigeon-pea.

5-3.1.3(b). Apparatus

The biolistic gun employs the principle of conservation of momentum and uses the passage of helium gas through the cylinder with arrange of velocities required for optimal transformation of various cell types. It consists of a bombardment chamber which is connected to an outlet for vacuum creation. The bombardment chamber consists of a plastic rupture disk below which macro carrier is loaded with micro carriers. These micro carriers consist of gold or tungsten micro pellets coated with DNA for transformation.

Figure 5-3.1.3(b). Working system of particle bombardment gun. The apparatus is placed in Laminar flow while working to maintain sterile conditions. The target cells/tissue is placed in the apparatus and a stopping screen is placed between the target cells and micro carrier assembly. The passage of high pressure helium ruptures the plastic rupture disk propelling the macro carrier and micro carriers.

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The stopping screen prevents the passage of macro projectiles but allows the DNA coated micro pellets to pass through it thereby, delivering DNA into the target cells.

5-3.1.3(c). Advantages

• Simple and convenient method involving coating DNA or RNA on to gold microcarrier, loading sample cartridges, pointing the nozzle and firing the device. • No need to obtain protoplast as the intact cell wall can be penetrated. • Manipulation of genome of sub-cellular organelles can be done. • Eliminates the use of potentially harmful viruses or toxic chemical treatment as gene delivery vehicle. • This device offers to place DNA or RNA exactly where it is needed into any organism.

5-3.1.3(d). Disadvantages

• The transformation efficiency may be lower than Agrobacterium- mediated transformation. • Specialized equipment is needed. Moreover the device and consumables are costly. • Associated cell damage can occur. • The target tissue should have regeneration capacity. • Random integration is also a concern. • Chances of multiple copy insertions could cause gene silencing.

5-3.1.4. Sonoporation

• Sonoporation involves the use of ultrasound for temporary permeabilization of the cell membrane allowing the uptake of DNA, drugs or other therapeutic compounds from the extracellular environment. • This method leaves the compound trapped inside the cell after ultrasound exposure.

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• It employs the acoustic cavitation of micro bubbles for enhancing the delivery of large molecules like DNA. The micro bubbles form complex with DNA followed by injection and ultrasound treatment to deliver DNA into the target cells. • Unlike other methods of transfection, sonoporation combines the capability to enhance gene and drug transfer.

Figure 5-3.1.4. Rupture of microbubbles by ultrasound resulting in enhanced membrane permeability caused by shear stress, increased temperature and activation of reactive oxygen species. Drug delivery by microbubbles by (a) transient holes induced by shear stress for drug delivery (b) increase in membrane fluidity (c) endocytosis of microbubbles (d) microbubble- cell membrane fusion. (Adapted fromhttp://88proof.com/synthetic_biology/blog/archives/192)

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5-3.1.4(a). Advantages

• Simple and highly efficient gene transfer method. • No significant damage is cause to the target tissue.

5-3.1.4(b). Disadvantages

• Not suitable for tissues with open or cavitated structures. • High exposure to low-frequency (

5-3.1.5. Laser induced transfection

• It involves the use of a brief pulse of focused laser beam. • In this method, DNA is mixed with the cells present in the culture and then a fine focus of laser beam is passed on the cell surface that forms a small pore sufficient for DNA uptake into the cells. The pore thus formed is transitory and repairs soon.

5-3.1.6. Bead transfection

• Bead transfection combines the principle of physically producing breaks in the cellular membrane using beads. • In this method, the adherent cells are incubated for a brief period with glass beads in a solution containing the DNA. • The efficiency of this rapid technique depends on:

o Concentration of DNA in a solution. o Timing of the addition of DNA. o Size and condition of the beads and the buffers utilized.

Immunoporation is a recently developed transfection process involving the use of new type of beads, ImmunofectTM beads, which can be targeted to make holes in a specific type of cells.

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Bibliography

Brown TA. 2006. Gene Cloning and DNA Analysis: an introduction. 5th ed. Blackwell Science Ltd.

Gehl J. 2003. Electroporation: theory and methods, perspectives for drug delivery, gene therapy and research. Acta Physiol Scand, 177: 437–447. http://88proof.com/synthetic_biology/blog/archives/192 http://www.bio.davidson.edu/courses/molbio/molstudents/ http://www.eplantscience.com http://www.nepadbiosafety.net/subjects/biotechnology/plant-transformation- bombardment

Kikkert JR, Vidal JR, Reisch BI. 2005. Stable transformation of plant cells by particle bombardment/biolistics. Methods Mol Biol.286: 61-78.

Mahamulkar S, Joshi V, Chavan A, Waghmare J, Waghmare S. 2010. Custom Made Animals The Magic of Transgenesis. Advanced Biotech Journal.

OhtaS, YukikoO, SuzukiK, KamimuraM, Tachibana K, Yamada G. 2011.Sonoporation for Gene Transfer into Embryos.Cold Spring HarbProtoc. doi:10.1101/pdb.prot5581

Primrose SB, Tyman RM, Old RW. 2001. Principle of Gene Manipulation. 6th ed. Wiley-Blackwell.

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Subject Name Botany Paper Name Plant Genetic Engineering Module Name/Title Choice of promoters for targeted expression of transgene, synthetic promoters Module Id Pre-requisites Basic knowledge about the promoters and plant genetic engineering Objectives To select appropriate promoter for expression of transgene at desired tissue or plant developmental stage and to understand synthetic promoters. Keywords Constitutivepromoters, Induciblepromoters, Organ/tissue specific promoters, synthetic promoters

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Choice of promoters for targeted expression of transgene, synthetic promoters

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Subject Coordinator Dr. Sujata Bhargava Savitribai Phule Pune University, Paper Coordinator Dr.Rohini Sreevathsa National Research Centre on Plant Biotechnology, Pusa, New Delhi Content Writer/Author Dr.Vitthal T. Barvkar Department of Botany, S. P. Pune (CW) University, Pune Content Reviewer (CR) Language Editor (LE)

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TABLE OF CONTENTS

1. Introduction 2. Types of Promoters 2.1 Constitutive 2.1.1 Virus derived constitutive promoters 2.1.2 Plasmid derived constitutive promoters 2.1.3 Plant derived constitutive promoters 2.2 Inducible 2.2.1 Promoters induced by abiotic stress 2.2.2 Promoters induced by biotic stress 2.2.3 Promoters induced by chemicals 2.3 Organ/tissue specific 3. Synthetic promoters

1. Introduction In an organism, gene expression is regulated at various stages like transcription, translation, post- translation. Transcription is an important step since it initiates gene expression. Promoter is a specific nucleotide sequence in DNA where RNA polymerase binds and starts transcription. Promoters are typically present upstream of the coding sequences. Promoter sequences are recognised by specific transcription factors which help in recruitment of RNA polymerase thus initiation of transcription. A typical promoter is made up of a core, proximal and distal promoter. Core promoter (located at approximately -34 bases upstream of Transcription start site (TSS)) consists ofRNA polymerase binding site, TSS and flanking sequences. Proximal(located at approximately -250 bases upstream of TSS) and distal promoter consists of various cis regulatory elements. These are mainly involved in tissue specific expression of target gene.

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Atypicalpromoter has complex arrangement ofmultiple clustered enhancer modules interspersed with silencer and insulator elementswhich can be located 10–50 kb either upstream or downstream of a composite corepromoter containing TATA box (TATA), Initiator sequences (INR), and downstreampromoter elements (DPE).(Adopted from Levine &Tjian, 2003)

In prokaryotes all the genes in one operon are driven by a single promoter. Prokaryotic promoter comprises of -10 and -35 consensus sequences. In eukaryotes each single protein coding gene has its own promoter and TATA box is the key regulatory element in core promoter. Promoters which show higher affinity with RNA polymerase and support high level of transcription are known as strong promoters (e.g. promoter of lac operon)while the promoters which show lower affinity with RNA polymerase and support low level of transcription are known as weak promoters (e.g. promoter of lac repressor gene) In genetic engineering, desired expression of transgene depends on the promoter used to drive the transgene hence promoter sequences are very important in transformation experiments. Large number of promoters in various plants have been characterized and are being used in developing transgenic plants.

2. Types of Promoters 2.1 Constitutive 2.2 Inducible 2.3 Organ/tissue specific 2.1 Constitutive promoters

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These are the most widely used promoters in plant transformation studies. Constitutive promoter confers high level of gene expression constantly in all the tissues and developmental stages and are largely, if not entirely, independent of environmental and developmental factors. A constitutive promoter contains an element which is recognised by basal transcription factors that are present in all the tissues.

http://www.bios.net/daisy/Bioindicators/g3/2117.html

Based on origin of cis acting element constitutive promoters have been categorized as: 2.1.1 Virus derived constitutive promoters 2.1.2 Plasmid derived constitutive promoters 2.1.3 Plant derived constitutive promoters

2.1.1Virus derived constitutive promoters: The Cauliflower mosaic virus 35S (CaMV 35S) promoter is the most widely used promoter in plant genetic transformations. CaMV 35S has been fused with number of genes involved in disease resistance, abiotic stress tolerance, modification of colour, scent etc. in various crops.This promoter can drive high level oftransgene expression in both dicots and monocotsThe major limitation ofvirus derived constitutive promoters is the abilityof plant cell to recognize thesesequencesas foreignandinactivatethem through methylationindependent and dependent manner and using chromatinremodeling.Other examples of virus derived constitutive promoters includeScBV (Sugarcane bacilliform virus),BSV (Banana streak badnavirus), CMPS

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(Cestrum Yellow Leaf curling virus), FMV (Figwort mosaic caulimovirus), MiMV (Mirabilis mosaic virus) etc. CaMV 19S promoter also can be used as a constitutive promoter.

2.1.2Plasmid derivedconstitutive promoters: Generally their origin is from Ti and Ri plasmids of Agrobacterium tumefaciens and Agrobacterium rhizogenesrespectively. As these are plant pathogen and some of the plasmid born genes are heavily transcribed by plant transcription machinery makes their promoters good candidates for driving constitutiveexpression in engineered plant. This includes promoters of nopaline synthase (nos), octopine synthase (ocs) and mannopine synthase (mas) genes.

2.1.3 Plant derived constitutive promoters: Genome sequencing of various plants enables us to find out sequences of promoters in plants. Generally these include housekeeping genes such as actin and ubiquitin gene promoters. These genes show high level of expression in plant tissues and their sequences are conserved. Examples of plant constitutive promoters include rice actin gene, ubiquitin promoter (Ubi) from maize and Arabidopsis, ubiquitin extension protein (uep) gene has been isolated from yeast and several plants, including tomato, barley and potato.

List of some constitutive promoters

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(Saranya&Kanchana, 2016) Advantages of constitutive promoters:  High level of expression of target gene in all tissues.  Helps in expression of selectable marker gene hence makes detection easy. Limitations of constitutive promoters:  Less effective in monocotyledons.  May get horizontally transferred and raise bio safety issues.

2.2 Inducible promoters: Genes regulated by inducible promoters are expressed in response to particular environmental stimulus. This occurs due to the activation of transcription factor that recognizes specific cis-acting sequences in inducible promoter. A single promoter can be induced by a combination of physical and chemical factors.

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Inducible promoters

Induced by Induced by physical agents chemical agents

e.g. Hormones, Antibiotic, Abiotic Factors Biotic factors Alcohol, Metals etc.

e.g. Light, Heat e.g. Pathogen shock, Cold attack, wounding

2.2.1 Promoters induced by abiotic stress:- These include the promoters induced by abiotic stresses such as salinity,drought, heat shock, cold, light etc.  Promoters of rice OsNCED3 and Wsi18 genes are drought inducible. These genes are involved in synthesis of ABA in rice. In transgenic rice with above promoters expression of target genes increased in response to drought and high salinity.  The Arabidopsis Rd29A promoter shows drought inducible expression of transgene when it was used in transgenic wheat.  The GLP promoter from Tamarixhispidais also a drought, salt and low temperature inducible promoter.  The potato proteinase inhibitor II gene (pinII) promoter is wound and UV irradiation inducible promoter. It has been used in transformation of Nicotianaplumbaginifolia and rice.

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 High or low temperatures, heavy metal or ABA induces the expression of Lehsp23.8 heat shock protein gene in tomato. Promoter of this gene has shown heat inducible expression of target gene in tobacco.  Sweet potato peroxides gene promoter (SWPA2) is induced by hydrogen peroxide and UV light.

2.2.2 Promoters induced by biotic stress:-  In transgenic apple and citrus plants Gst promoter (isolated from potato) shows transcription of target after bacterial and fungal attack.  Use of pathogen inducible promoter PR10 in grapes induces expression of plant defense gene on fungal attack.  Apart from these examples various pathogen inducible promoters have been characterized in different plants.

(Gurr&Rushton, 2005)

Mechanism of inducible promoters

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2.2.3 Promoters induced by chemicals:- Chemically inducible promoters include alcohol regulated promoters, tetracycline regulated promoters, hormone responsive promoters, metal-responsive promoters.

E.g. IAA4/5 gene promoter(Auxin inducible), 4CL promoter (Jasmonates and alpha-linolenic acid inducible),alcA promoter(Alcohol inducible), PR-1a tobacco promoter (2,6-dichloroisonicotinic acid inducible) etc.

Inducible promoters can be turned on or off by manipulating environmental stimulus hence they are preferred over constitutive promoters. Most of the inducible promoters are activated quickly after generation of stimulus and does not interfere with normal development processes. However, some endogenous promoters create low level of expression which limits the use of inducible promoter.

List of some Inducible promoters

(Saranya&Kanchana, 2016)

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2.3 Tissue/Organ specific promoters:- Constitutive promoters are not useful in obtaining transgene expression in specific tissues. Moreover constitutive expression can interfere in normal plant functioning. Hence there is a need of tissue/organ specific expression. Tissue specific promoters fulfill this need of targeted expression. Number of plant organ/tissue/developmental stage specific promoters have been characterized in different plants.

 Fruit specific promoters: Ethylene is a major hormone involved in fruit ripening. Promoters of ACC oxidase, E4, E8, Polgalactouronase gene have been well studied in tomato and apple. They also have commercial application in manipulating fruit ripening. Strawberry gene Faxy1 promoter, banana sucrose phosphate synthase (SPS) promoter, Citrus C11 promoter are some other examples of fruit specific promoter.

 Seed specific promoters: Seeds are the major part of human diet. They are the storage organs of plants. Seeds act as a reservoir of proteins and serves as nitrogen source for developing embryo. Promoters of these seed storage proteins have been characterized to achieve seed specific expression of transgene.

E.g.Rice glutelin and globulin, endosperm-specific hordein promoters in barley, glutenin promoters from wheat, soya lectin and β-phaseolin promoters are well characterized.

 Root specific promoters: Several promoters of root specific genes involved in absorption, nutrient uptake, nodule formation, nematode resistance, storage etc. have been identified. These include RB7 gene from tobacco(membrane channel aquaporin), French bean gln gamma gene promoter(nodule formation), class I patatin(storage) etc.

 Floral organ specific promoters: Targeted gene expression in floral organs can lead to improvement in colour, fragrance, vase life etc. Chalcone synthase (CHS) gene is involved in biosynthesis of flavonoides and other secondary metabolites, pigments such as anthocyanins, insect attractant or repellent, phytoalexins etc. Promoter of CHS is well studied in Phaseolus, Antirrhinum

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andPetunia.Other floral organ specific promoters are pistil-specific thaumatin/ PR5-like protein (PsTL1) promoter from pear, LAT52 and LAT59 anther-specific gene promoters from tomato etc.

 Vascular tissue specific promoters: Tissue specific expression in conducting tissue (xylem & phloem) at the site of infection provides resistance against aphids, wounding etc. Xylem specific promoter (PAL2), Phloem specific promoters (rolC,CoYMV) have been identified.

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3. Synthetic promoters:- In addition to above promoters, there is a need of promoter which can precisely regulate transgene expression. With the help of synthetic promoters multiple transgenes can be successfully incorporated into plant system.

Synthetic promoters consists of a core promoter or minimal promoter often derived from CaMV 35S promoter sequences. It consists of TATA box, 50 bp away from Transcription Start Site (TSS) and additional sequences such as CAAT box etc.

Along with core promoter, one or more synthetic motifs are present in synthetic promoter. These synthetic motifs can be selected from databases like TRANSFAC or can be generated de novo using bioinformatics. Number, type and spacing between synthetic motifs determines the regulated expression of target genes. Some of the synthetic promoters are listed in the table:

From: (Wusheng&Stewat, 2016)

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SUGGESTED READING 1.S.B. Primrose and R.M. TwymanPrinciples of Gene Manipulation and Genomics 2006 Blackwell Publishing Seventh edition

2. DuttM. et. al. 2014 Temporal and spatial control of gene expression in horticultural crops Nature Horticulture Research (2014) 1, 14047; doi:10.1038/hortres.2014.47

3. Liu W and Stewart CN Jr.Plant synthetic promoters and transcription factorsCurrent Opinion in Biotechnology 2016 37:36-44. doi: 10.1016/j.copbio.2015.10.001.

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