Gene Transfer Methods • The delivery of DNA into the host is required for generation of genetically modified organism. • DNA delivery to host is a 3 stage process, DNA sticking to the host cell, internalization and release into the host cell. • As a result, it depends on 2 parameters- • Surface chemistry of host cell-Host cell surface charges either will attract or repel DNA as a result of opposite or similar charges. Presence of cell wall (in the case of bacteria, fungus and plant) causes additional physical barrier to the uptake and entry of DNA. • Charges on DNA-Negative charge on DNA modulates interaction with the host cell especially cell surface. DNA transfer by natural methods

1. Conjugation 2. Bacterial transformation 3. Retroviral 4. Agrobacterium mediated transfer Conjugation

• Requires the presence of a special plasmid called the F plasmid. • Bacteria that have a F plasmid are referred to as as F+ or male. Those that do not have an F plasmid are F- or female. • The F plasmid consists of 25 genes that mostly code for production of sex pilli. • A conjugation event occurs when the male cell extends its sex pilli and one attaches to the female. • This attached pilus is a temporary cytoplasmic bridge through which a replicating F plasmid is transferred from the male to the female. • When transfer is complete, the result is two male cells. • When the F+ plasmid is integrated within the bacterial chromosome, the cell is called an Hfr cell (high frequency of recombination cell).

TRANSFORMATION

• Transformation is the direct uptake of exogenous DNA from its surroundings and taken up through the . • Transformation occurs naturally in some species of bacteria, but it can also be effected by artificial treatment in other species. • Cells that have undergone this treatment are said to be competent.

TRANSDUCTION

• Gene transfer from a donor to a recipient by way of a . • If the lysogenic cycle is adopted, the phage chromosome is integrated (by covalent bonds) into the bacterial chromosome, where it can remain dormant for thousands of generation • The lytic cycle leads to the production of new phage particles which are released by lysis of the host.

AGROBACTERIUM MEDIATED TRANSFER

• Agrobacterium tumefaciens is a soil borne gram negative bacterium. • It invades many dicot plants when they are injured at the soil level and causes crown gall disease. • The ability to cause crown gall disease is associated with the presence of the Ti (tumour inducing) plasmid within the bacterial cell. • Ti plasmid can be used to transport new genes into plant cells. THE Ti-PLASMIDS

• A remarkable feature of the Ti plasmid is that, after infection, part of the molecule is integrated into the plant chromosomal DNA . • This segment, called the T-DNA, is between 15 and 30 kb in size, depending on the strain. • T-DNA contains eight or so genes that are expressed in the plant cell and are responsible for the cancerous properties of the transformed cells. • These genes also direct synthesis of unusual compounds, called opines, that the bacteria use as nutrient. • The vir (virulence) region of the Ti- plasmid contains the genes required for the T-DNA transfer process. • The genes in this region encode the DNA processing enzymes required for excision, transfer and integration of the T-DNA segment. • The T-DNA region of any Ti plasmid is defined by the presence of the right and the left border sequences. • These border sequences are 24 bp imperfect repeats. • Any DNA between the borders will be transferred in to the genome of the plant. Ti-Plasmid mediated transfer of gene into a plant

• The Ti-Plasmid has an innate ability to transmit bacterial DNA into plant cells. • The gene of a donor organism can be introduced into the Ti plasmid at the T-DNA region • This plasmid now becomes a recombinant plasmid. • By Agrobacterium infection, the donor genes can transferred from the recombinant Ti- Plasmid and integrated into the genotype of the host plant. DNA TRANSFER BY ARTIFICIAL METHODS

• Physical methods – Microinjection – Biolistics transformation • Chemical methods – DNA transfer by calcium phosphate method – Liposome mediated transfer • Electrical methods – Electroporation Electroporation

• Electroporation uses electrical pulse to produce transient pores in the plasma membrane thereby allowing DNA into the cells. • These pores are known as electropores. • The phospholipid molecules of the plasma membrane are not static. • When we apply elec tric field to them their kinetic energy increases resulting in the increase in the membrane per meability at certain points. • This is exactly where we see the formation of electro- pores. • The cells are mixed with the recom binant DNA and the mixture is placed in a small chamber with electrodes connected to a specialized power supply. • A brief electric impulse is discharged across the elec trodes, which makes pores (holes) in the plasma membrane. • These pores remain for sometime and are again resealed themselves. • Recombinant DNA enters the cell which are removed and plated in fresh selective medium. • The process of selection is then applied to iso late cells carrying recombinant DNA. Advantages of Electroporation

1. Method is fast. 2. Less costly. 3. Applied for a number of cell types. 4. Simultaneously a large number of cell can be treated. 5. High percentage of stable transformants can be produced Microinjection • The microinjection is the process of transferring the desirable DNA into the living cell ,through the use of glass micropipette. • In this procedure the cell is held on a glass capillary by gentle suction. • Using a micromanipulator (a mechanical device for fine control of the capillary) the needle has been inserted into the nucleus of the host cell. • Glass micropipette is usually of 0.5 to 5 micrometer, easily penetrates into the cell membrane and nuclear envelope. • The desired gene is then injected into the sub cellular compartment and needle is removed

Limitations of microinjection

• Costly. • Skilled personal required/ labour-intensive Biolistics or Microprojectiles

• Biolistics or particle bombardment is a physical method that uses accelerated microprojectiles to deliver DNA or other molecules into intact tissues and cells. • The gene gun is a device that literally fires DNA into target cells . • The DNA to be transformed into the cells is coated onto microscopic beads made of either gold or tungsten. • The coated beads are then attached to the end of the plastic bullet and loaded into the firing chamber of the gene gun. • An explosive force fires the bullet with DNA coated beads towards the target cells that lie just beyond the end of the barrel. • Some of the beads pass through the cell wall into the cytoplasm of the target cells

Sonoporation • Sonoporation, or cellular sonication, is the use of sound (typically ultrasonic frequencies) for the transfer of recombinant DNA into the target host cell. • This process has been success fully used in a wide range of host cells starti ng from bacteria to plant and animal cells. • This employs the acoustic waves to increase the permeability of the plasma membrane. • Taking the advantage of this situation the recombinant DNA enters the host cell. Optical

• Optical Transfection is the process of introduc ing nucleic acids into cells using light. • This has been successful in transfecting animal cells. • In this technique the plasma membrane of the host cell is exposed to the highly focused la ser beam for a small amount of time (typically tens of milliseconds to seconds), generating a transient pore on the membrane called photo-pore. • Through the photo-pore the recombinant DNA can enter the host cell. Silicon carbide fibers mediated • SCF of 0.6µm in diameter and 10-80µm in length are used. • Mechanism of DNA transfer is penetration. Calcium Chloride (CaCl2) Mediated DNA Transfer • All bacterial cells cannot uptake the exogenous DNA mole cule. • Cells capable to take DNA are called competent cells. • Bacterial cells are made more competent so that the possibility of transferring of the recombi nant DNA into the host cell increases to a higher fold.

• CaCl2 makes the cell wall of the bacteria more permeable to the exogenous DNA and thus increases the competence of the host cell.

Calcium phosphate mediated DNA transfer

• The process of transfection involves the mixture of isolated DNA (10-100ug) with solution of calcium chloride and potassium phosphate so precipitate of calcium phosphate to be formed. • Cells are then incubated with precipitated DNA either in solution or in tissue culture dish. • A fraction of cells will take up the calcium phosphate DNA precipitate by endocytosis.

Polyplexes method • The disadvantage of calcium phosphate method is severe physical damage to the cellular integrity due to particulate matter settling on the cell. • It results in reduced cellular viability and cytotoxicity to the cell. • An alternate method was evolved where DNA was complexed with chemical agent to form soluble precipitate (polyplexes- polycationic compounds that form soluble complexes) through electrostatic interaction with DNA. • A number of polycationic carbohydrate (DEAE-Dextran), positively charged cationic lipids (transfectin), polyamines (polyethylenimines) etc are used for this purpose. • The soluble aggregates of DNA with polycationic complex adhere to the cell membrane and enter into the cytoplasm via endocytosis or osmotic shock induced by DMSO or glycerol and it reaches to the nucleus for expression Protoplast Fusion

• This technique is used for introducing gene of interest into plant and animal cells. • In this technique first we transfer the recombinant DNA into a bacterial cell then dissolve its cell wall by treating it with lysozyme. • After this we fuse the host protoplast with the bacterial cell (lacking cell wall) by the help of polyethy lene glycol (PEG). • The transfected cells are then selected by suitable methods. Polyethylene glycol mediated transfection

• This method is utilized for protoplast only. • Polyethylene glycol stimulates endocytosis and therefore DNA uptake occurs. • Protoplasts are kept in the solution containing polyethylene glycol (PEG). • After transfer of DNA to the protoplast in presence of PEG and other chemicals, PEG is allowed to get removed Lithium Acetate/ssDNA/PEG Method

• In this method, yeast cells are incubated with a transformation mixture of lithium acetate, PEG, single stranded carrier DNA and foreign plasmid at 420C for 40mins. • LiAc and heat shock – Helps DNA to pass through the cell wall • The carrier DNA is added to block the non-specific sites on cell wall and made plasmid available for uptake. • Post-transformation, cells are pelleted to remove transformation mixture and re-suspended in 1ml water. • It is plated on a solid media with an appropriate selection pressure such as antibiotics. Spheroplast Transformation Method

• Yeast cell wall is removed partially to produce spheroplast. • Spheroplasts are very fragile for osmotic shock but are competent to take up free DNA at high rate. • In addition, polyethyl glycol (PEG) is used to facilitate deposition of plasmid and carrier DNA on cell wall for easier uptake. • The mechanism of DNA uptake in yeast is not very clear.

Liposome and lipoplex method

• Another approach of DNA transfection in animal cell si to pack the DNA in a lipid vesicle or liposome. • In this approach, DNA containing vesicle will be fused with the cell membrane and deliver the DNA to the target cell. • Preparation of liposome and encapsulating DNA was a crucial step to achieve good transfection efficiency. • Liposome prepared with the cationic or neutral lipid facilitates DNA binding to form complex (lipoplex) and allow uptake of these complexes by endocytosis. • The lipoplex method was applicable to a wide variety of cells, and found to transfect large size DNA as well. • Another advantage of liposome/lipoplexes is that with the addition of ligand in the lipid bilayer, it can be used to target specific organ in the animal or a site within an organ. Advantages

• Simplicity. • Long term stability. • Low toxicity. • Protection of nucleic acid from degradation Bactofection

• This mode of gene transfer is very popular in plant where agrobacterium tumefaciens is used. • In animal cell, bacteria is actively been taken up by the host cell through phagocytosis and entrapped in a membraneous vesicle known as phagosome. • Then bacteria get escape from phagosome and get lysed to release the DNA into the cytosol. • In alternate mechanism, bacteria get lysed inside the phagosome and DNA is released into the cytosol. • The bacterial species used in this methods are salmonella, shigella etc. • Most of the strain used to deliver the DNA are attenuated so that they should not harm host cell Mechanism of transfection of mammalian cell with bacteria Transduction ( mediated)

• Viral particle has a natural tendency to attack and deliver the DNA into the eukaryotic cells. • gene of interest in to the viral vectors is a innovative way to deliver the DNA into the host cell. • If the recombination sequences are available, the delivered DNA is integrated into the host and replicate. • Virus has essential components for expression of proteins required for DNA replication, RNA polymerase and other ligand for attachment onto the host cell. • In addition, it has additional structural components to regulate infection cycle. • The virus vector contains cassettes to perform all these functions then it is fully sufficient to propagate independently. • Few virus strains may cause disease if their propagation will be uncontrolled. • A mechanism has been devised to keep a check on the uncontrolled propagation of virus in cell. • Few crucial structural blocks are placed on another helper plasmid, in this case virus propagate only if helper plasmid has been supplied along with the viral vector. • This particular arrangement is made with the virus strains which can cause disease after integrating into the genome.