An Efficient Method for Isolation of Plasmid DNA for Transfection Of

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Protocol An Eﬃcient Method for Isolation of Plasmid DNA for Transfection of Mammalian Cell Cultures

1,2, , 3, 3 Daniel V. Kachkin * † , Julia I. Khorolskaya †, Julia S. Ivanova and Aleksandr A. Rubel 1,2,* 1 Laboratory of Amyloid Biology, St. Petersburg State University, 199034 St. Petersburg, Russia 2 Department of Genetics and Biotechnology, St. Petersburg State University, 199034 St. Petersburg, Russia 3 Institute of Cytology Russian Academy of Science, 194064 St. Petersburg, Russia; [email protected] (J.I.K.); [email protected] (J.S.I.) * Correspondence: [email protected] (D.V.K.); [email protected] (A.A.R.); Tel.: +7-9111333968 (D.V.K.); +7-9111333968 (A.A.R.) These authors contributed equally to this work. † Received: 1 September 2020; Accepted: 1 October 2020; Published: 14 October 2020

Abstract: In this article, we present several protocols that describe the steps from cloning and obtaining a large amount of pure plasmid DNA to generation of lentiviruses based on these constructs. The protocols have been worked out on human cell culture HEK293T but can be adapted for other cell cultures. This protocol was designed to be simple to execute and cheap since it requires only materials and consumables widely available in molecular laboratories, such as salts, alcohols, etc., and no complicated laboratory equipment. These protocols are highly eﬀective and can be performed in any standard molecular biology laboratory.

Keywords: plasmid; mammalian cell transfection; lentiviral transduction; lentivirus; HEK293T; E. coli

1. Introduction Transfection is a procedure that introduces DNA and RNA into eukaryotic cells to produce genetically modified cells. Transfection is a powerful analytical tool for studying gene function and their regulation by enhancing or inhibiting specific gene expression in cells. It is also important for studying proteins or producing recombinant proteins in mammalian cells [1]. Transfection can be used in gene therapy, for production of recombinant proteins for therapeutic purposes, small interference RNA knockdown procedures [2] and is an essential step for lentivirus production [3]. There are various approaches for mammalian cell transfection. However, one of the key factors for successful transfection remains the quality and the quantity of the transfected material (DNA, RNA). To transfect human cells, a researcher needs a large amount of high-quality DNA. DNA midi- or maxiprep kits are often expensive and the amount of DNA isolated with their help is often insufficient. In this study, we developed a new protocol suitable to achieve a high yield of plasmid DNA of high purity, acceptable for the transfection of mammalian cell cultures. Our method can also significantly reduce the cost of DNA isolation, since it provides a high yield of extracted plasmid without an implementation of commercial kits. For this method, we used the E. coli bacterial strain STBL3 (ThermoFisher Scientific, Walthon, MA, USA). This strain was designed especially for cloning the direct repeats found in lentiviral expression vectors and it gives a higher yield of extracted DNA. However, the introduced protocol is probably compatible with any E. coli strain used for the cloning. The DNA purity is sufficient for basic transfection methods using cationic polymers, such as PEI (polyethylenimine) or TurboFect (ThermoFisher, Walthon, MA, USA). Transfection using PEI has several advantages, like its cheapness and suitability for various cell cultures [4].

Methods Protoc. 2020, 3, 69; doi:10.3390/mps3040069 www.mdpi.com/journal/mps Methods Protoc. 2020, 3, x FOR PEER REVIEW 2 of 9

Lentiviral technology came to widespread use in molecular and cell biology for stable gene expression, gene silencing, generating transgene animals, induction of pluripotent stem cells, stem cell modification and lineage tracking, immunization and in vivo imaging [5]. Lentiviral technology is based on the co-transfection of human cell line HEK293T with packaging, envelope and transfer plasmids (containing the gene of interest). This is resulted in the assembly of the lentivirus, which, upon transduction, became a powerful tool for the expression of exogenous genes into various types of cells both in vitro and in vivo. Today, this method is the most effective way to obtain stable cell linesMethods producing Protoc. 2020 the, 3, studied 69 genetic products [6]. 2 of 9 These protocols can be easily reproduced in almost any lab, they have a good efficiency and may be necessary for the wide range of studies in cell and molecular biology. Lentiviral technology came to widespread use in molecular and cell biology for stable gene In this article, we offer well-reproducible, high-performance protocols that are guided and expression, gene silencing, generating transgene animals, induction of pluripotent stem cells, stem cell adapted to work with mammalian cell cultures. This protocol of plasmid DNA extraction is also modification and lineage tracking, immunization and in vivo imaging [5]. Lentiviral technology is applicable to other techniques where a large amount of plasmid DNA is required. based on the co-transfection of human cell line HEK293T with packaging, envelope and transfer plasmids (containing the gene of interest). This is resulted in the assembly of the lentivirus, which, 2. Experimental Design upon transduction, became a powerful tool for the expression of exogenous genes into various types of cellsIn both this inarticle, vitro andwe proposein vivo. an Today, optimized this method protoc isol the for most the isolation effective wayof plasmid to obtain DNA stable (Figure cell lines 1) fromproducing bacterial the cultures studied without genetic productsthe use of [6 commercial]. kits for its subsequent application in genetic modificationsThese protocols of mammalian can be easily cell cultures. reproduced The in advantages almost any of lab, this they method have a goodare as e ffifollows:ciency anda high may efficiencybe necessary (concentration for the wide of rangeplasmi ofd studiesDNA at in the cell output and molecular is 3–8 mg/mL), biology. availability, sufficient purity of the Inobtained this article, material, we off aser well-reproducible,well as cheapness high-performancealong with relatively protocols low time that costs. are guided In addition, and adapted the protocolto work contains with mammalian a description cell cultures.of the methods This protocol for efficient of plasmid transfection DNA extraction and assembly is also of applicable lentiviral to particlesother techniques based on the where obtained a large plasmid amount DNA, of plasmid which DNA have isbeen required. successfully tested for immortalized and primary mammalian cell cultures. 2. ExperimentalWe recommend Design using plasmids containing fluorescent labels, as this will provide the best controlIn over this the article, experiment, we propose calculation an optimized of transfection/transduction protocol for the isolation efficiency of plasmid and viral DNA assembly. (Figure 1) fromThe bacterial plasmids cultures obtained without for the usestudy of commercialwere transformed kits for into its subsequent E. coli according application to the in Inoue genetic standardmodifications protocol of [7]. mammalian We used the cell plasmids cultures. pLenti-CMV-EGFP The advantages of Hygro this method (656-4) are[8], aspMD2 follows: (gift afrom high Didierefficiency Trono (concentration (Addgene plasmid of plasmid # 12259)) DNA and at the PAX2 output (gift is from 3–8 mg Didier/mL), Trono availability, (Addgene suffi cientplasmid purity # 12260)).of the obtainedThe protocol material, is based as well on as the cheapness Birnboim along and with Doly relatively DNA lowextraction time costs. protocols In addition, with modificationsthe protocol contains[9,10]. a description of the methods for efficient transfection and assembly of lentiviral particlesThis protocol based on lists the the obtained plasmids plasmid and strain DNA, used which in have this study, been successfully but the DNA tested extraction for immortalized protocol isand suitable primary for all mammalian strains and cell plasmids. cultures. It is suitable for all researchers working with cell cultures due to its simplicity and it does not require complicated equipment or expensive reagents.

FigureFigure 1. 1.ExperimentalExperimental design. design. Scheme Scheme of (A of) the (A) experiment the experiment for the for is theolation isolation of plasmid of plasmid DNA; DNA; (B) the(B )experiment the experiment for the for mammalian the mammalian cells cells transfection; transfection; (C) (theC) theexperiment experiment for for the the lentiviral lentiviral vector vector production.production. All All stages stages after after the the change change of ofmedia media on on Day Day 4 should 4 should be be carried carried out out with with the the proper proper biosafetybiosafety containment recommendedrecommended for researchfor research with lentiviralwith lentiviral vectors. Createdvectors. with Created BioRender.com. with BioRender.com. We recommend using plasmids containing fluorescent labels, as this will provide the best control over the experiment, calculation of transfection/transduction efficiency and viral assembly. The plasmids obtained for the study were transformed into E. coli according to the Inoue standard protocol [7]. We used the plasmids pLenti-CMV-EGFP Hygro (656-4) [8], pMD2 (gift from Didier Trono (Addgene plasmid # 12259)) and PAX2 (gift from Didier Trono (Addgene plasmid # 12260)). The protocol is based on the Birnboim and Doly DNA extraction protocols with modifications [9,10]. Methods Protoc. 2020, 3, 69 3 of 9

This protocol lists the plasmids and strain used in this study, but the DNA extraction protocol is suitable for all strains and plasmids. It is suitable for all researchers working with cell cultures due to its simplicity and it does not require complicated equipment or expensive reagents.

2.1. Materials Tris base (ThermoFisher Scientific, USA; Cat. no.:15504020); • EDTA Disodium Salt 2-hydrate (PanReac AppliChem, USA; Cat. no.:141669); • Yeast Extract w/o salts (Helicon, Russia; Cat. no.: H-0601MG-0.5); • D-Glucose monohydrate (Helicon, Russia; Cat. no.: Roquette-361103-0.5); • Tryptone (VWR Life Science AMRESCO, USA; Cat. no.: 97063-388); • Agar, European type (PanReac AppliChem, USA; Cat. no.: 402302.1210-1); • Sodium Cloride (Helicon, Russia; Cat. no.: H-1418-1.0); • Lithium Chloride (Sigma-Aldrich, USA; Cat. no: L4408); • Sodium Acetate (PanReac AppliChem, USA; Cat. no.: 131633); • Sodium hydroxide (PanReac AppliChem, USA; Cat. no.: 211687); • Sodium Dodecyl Sulfate (PanReac AppliChem, USA; Cat. no.: 142363); • RNAse A (Sigma-Aldrich, USA; Cat. no: R6148); • Antibiotic for plasmid selection (we used Ampicillin (Sigma-Aldrich, USA; Cat. no.: 10835242001)); • Ethanol; • Isopropanol; • Dulbecco’s Modified Eagle Medium (DMEM; Gibco, USA; Cat. no.: 41965039); • Opti-MEM Reduced-Serum Medium (Gibco, USA; Cat. no.: 51985026); • Defined Fetal Bovine Serum, (FBS; HyClone, USA; Cat. no.: SH30070.03); • L-glutamine (Gibco, USA; Cat. no.:25030024); • Penicillin-streptomycin (Gibco, USA; Cat. no.:15070063); • Polyethylenimine (PEI) HCl MAX, MW 40000 (Polysciences, USA; Cat. no.: 24765-1); • Hexadimethrine bromide (Polybrene) (Sigma-Aldrich, USA; Cat. no.: 107689); • Bacterial strain STBL3 (ThermoFisher Scientific, USA); • Human cell line HEK293T (Cells were obtained from the Russian Cell Culture Collection (Institute of • Cytology, St. Petersburg, Russia)); pMD2—envelope plasmid (gift from Didier Trono, addgene plasmid # 12259); • PAX2—packaging plasmid (gift from Didier Trono, addgene plasmid # 12260); • pLenti-CMV-GFP Hygro (656-4)—transfer lentiviral plasmid [8]. • 2.2. Equipment Tissue culture-treated plates (TPP Techno Plastic Products AG Schaffhausen, Trasadingen, Switzerland); • 15 mL Falcon Conical Centrifuge Tubes (Corning, Corning, USA; Cat. no.: 352095); • 50 mL Conical Centrifuge Tubes (Corning, Corning, NY, USA; Cat. no: 352070); • Syringe 0.45-µm filter (Jet Bio-Filtration, Guangzhou, China; Cat. no.: FPE404030); • Polycarbonate centrifuge bottles (Beckman Coulter, Brea, CA, USA; Cat. no: 363420); • New Brunswick Galaxy 170R CO incubator (Eppendorf, Hamburg, Germany); • 2 Class II, Type A2 Biological Safety Cabinets (Lamsystems, Miass, Russia); • ZOE Fluorescent cell imager (Bio-Rad, Hercules, CA, USA); • Inverted microscope Eclipse TS100 (Nikon, Tokyo, Japan); • Vortex Genius 3 (IKA, Staufen, Germany); • Shaker incubator (Biosan, Riga, Latvia); • Petri dishes for microbiology (Helicon, Moscow, Russia); • Methods Protoc. 2020, 3, 69 4 of 9

Centrifuge with cooling Eppendorf 5810 R (Eppendorf, Hamburg, Germany); • Avanti J-E Centrifuge (Beckman Coulter, Brea, CA, USA; Cat. no: 369005); • CytoFLEX Flow Cytometer (Beckman Coulter, Brea, CA, USA); • Spectrophotometer Nanodrop TB-1000, ThermoFisher Scientiﬁc, Walthon, MA, USA). • 3. Procedure

3.1.Methods Plasmid Protoc. 2020 DNA, 3, xIsolation FOR PEER fromREVIEW E. coli Bacteria—Time for Completion: 16–18 h 4 of 9 1. Inoculate a single colony transformed with the desired plasmid from the plate with LB (+ antibiotic 3. Procedure for selection) into 50 mL of liquid medium (LB or TBR) with the appropriate antibiotic added. 3.1. PlasmidNote: DNA The ﬂaskIsolation for from growing E. coli the Bacteria—Time bacteria should for Completion: be at least 16–18 4–5 timesh larger than the volume of the medium in which the bacteria grow. This is necessary for a better aeration of the culture. 1. Inoculate a single colony transformed with the desired plasmid from the plate with LB (+ 2. antibioticGrow the for cells selection) with vigorousinto 50 mL shaking of liquid overnightmedium (LB at or 37 TBR)◦C. with the appropriate antibiotic 3. added.Transfer the cells to centrifuge tubes (V = 50 mL) and centrifuge at 4000 g for 10 min at × Note:room The temperature. flask for growing the bacteria should be at least 4–5 times larger than the volume of 4. theCarefully medium drain in which the supernatantthe bacteria grow. and resuspendThis is necessary the cell for pellet a better in aeration 5 mL of of sterile the culture. water or TEG with 2. Growthe addition the cells with of RNAse vigorous (3–5 shakingµL/mL). overnight Add TEG at 37 (Tris-EDTA-Glucose °C. buﬀer) at sterile conditions,

3. Transferto avoid the contamination. cells to centrifuge tubes (V = 50 mL) and centrifuge at 4000 g for 10 min at room temperature. 5. Add 10 mL of a lysis solution (0.2N NaOH; 1% SDS) to the cell suspension. Gently mix by 4. Carefully drain the supernatant and resuspend the cell pellet in 5 mL of sterile water or TEG withinverting the addition the tube of untilRNAse all cells(3–5 μ areL/mL). lysed. Add It isTEG important (Tris-EDTA-Glucose to mix the tube buffer) gently at sterile by inverting to conditions,avoid contamination to avoid contamination. by genomic DNA. 6.5. AddAdd 10 7.5 mL mL of of a 3Mlysis sodium solution acetate (0.2N (pHNaOH;= 5.0) 1% andSDS) mix to thoroughly.the cell suspension. Incubate Gently the sample mix by for 10 min invertingat 4 ◦C. the tube until all cells are lysed. It is important to mix the tube gently by inverting to avoidNote: contamination If the precipitate by genomic does not DNA. settle well or flocs remain in the solution after the centrifugation, 6. Add 7.5 mL of 3M sodium acetate (pH = 5.0) and mix thoroughly. Incubate the sample for then the volume of the sodium acetate solution may be increased up to 10 mL or its pH is brought 10 min at 4 °C. Note:to 4.8. If the precipitate does not settle well or flocs remain in the solution after the centrifugation, 7. Centrifuge the sample for 15 min at 4000 g at 4 C. then the volume of the sodium acetate solution× may◦ be increased up to 10 mL or its pH is 8. Transferbrought the to supernatant 4.8. that contains the plasmid DNA to a new tube and add an equal volume 7. Centrifugeof isopropanol. the sample Thoroughly for 15 min mix at 4000 and g incubateat 4 °C. the sample for 15 min at room temperature. It is 8. Transferimportant the supernatant to use high that quality contains isopropanol the plasmid as DNA this directly to a new atubeffects and the add purity an equal of the volume DNA. of isopropanol. Thoroughly mix and incubate the sample for 15 min at room temperature. It is 9. Centrifuge for 20 min at 4000 g at room temperature. important to use high quality isopropanol× as this directly affects the purity of the DNA. 10.9. CentrifugeDrain the for supernatant 20 min at 4000 and g dryat room the precipitate.temperature. Dissolve the precipitate in 1 mL of sterile water. 11. Add 1 mL of 9M LiCl to the sample, mix thoroughly and incubate 20 min at 20 ◦C. 10. Drain the supernatant and dry the precipitate. Dissolve the precipitate in 1 mL of sterile− water. 12.11. AddCentrifuge 1 mL of 9M for LiCl 20 min to the at 4000sample,g mixat th4oroughlyC. and incubate 20 min at - 20 °C. × − ◦ 13.12. CentrifugeCarefully for transfer 20 min the at 4000 supernatant g at - 4 °C. to a new centrifuge tube (V = 15 mL). Discard the precipitate 13. Carefully(it contains transfer RNA the and supernatant protein residues). to a new centrifuge tube (V = 15 mL). Discard the precipitate (it contains RNA and protein residues). 14. Add 5 mL of 96% ethanol to the supernatant. Vortex thoroughly. 14. Add 5 mL of 96% ethanol to the supernatant. Vortex thoroughly. 15. Incubate for 1 h at 20 C. 15. Incubate for 1 h at −20− °C. ◦ PAUSE STEP STEP It Itis ispossible possible to pause to pause the protocol the protocol here and here leave and samples leave samples at −20 °C at overnight.20 ◦C overnight. 16. Centrifuge at 4000 g for 20 min at 4 °C. − 16. Centrifuge at 4000 g for 20 min at 4 ◦C. 17. Discard the supernatant× and thoroughly dry the precipitate. 17. Discard the supernatant and thoroughly dry the precipitate. 18. Dissolve the plasmid DNA in 500 μL of sterile water or TE buffer. 18. Dissolve the plasmid DNA in 500 µL of sterile water or TE buffer. 3.2. Transfection of Human Cell Culture HEK293T Using PEI—Time for Completion: 3–5 Days 3.2. Transfection of Human Cell Culture HEK293T Using PEI—Time for Completion: 3–5 Days 1. One day before the transfection, seed HEK293T cells into a multi-well culture plate or a tissue culture 1. dishOne with day respect before to the the transfection, required number seed of HEK293Tcells per 1 cm cells2 (the into approximate a multi-well number culture of cells plate for or a tissue differentculture culture dish with dishes respect is shown to in the Table required 1). Cultivate number the cells of cells overnight per 1 in cm DMEM2 (the supplemented approximate number withof cells for10% di fferentFBS, culture 1% dishesL-glutamine, is shown in Tableand 1). Cultivate1% penicillin/streptomycin. the cells overnight in DMEM supplemented CRITICAL STEP with It is 10% crucial FBS, to 1% check L-glutamine, the cells for mycoplasma and 1% penicillin contamination;/streptomycin. the presence of mycoplasma may significantly reduce the efficiency of the transfection or transduction. 2. Next day right before transfection prepare the DNA and PEI solution (transfection mix). The volume of transfection mix is 10% of the total volume of the culture medium (recommended by the manufacturer of culture dishes). Add the required amount of plasmid DNA (see Table 1) to the Opti- MEM and mix well on a vortex. Then, dropwise add PEI (1 mg/mL) to the DNA solution while vortexing. Note: We recommend using plasmid DNA in a final concentration 1–2 mg/mL. The amount of PEI Methods Protoc. 2020, 3, x FOR PEER REVIEW 4 of 9

3. Procedure

3.1. Plasmid DNA Isolation from E. coli Bacteria—Time for Completion: 16–18 h 1. Inoculate a single colony transformed with the desired plasmid from the plate with LB (+ antibiotic for selection) into 50 mL of liquid medium (LB or TBR) with the appropriate antibiotic added. Note: The flask for growing the bacteria should be at least 4–5 times larger than the volume of the medium in which the bacteria grow. This is necessary for a better aeration of the culture. 2. Grow the cells with vigorous shaking overnight at 37 °C. 3. Transfer the cells to centrifuge tubes (V = 50 mL) and centrifuge at 4000 g for 10 min at room temperature. 4. Carefully drain the supernatant and resuspend the cell pellet in 5 mL of sterile water or TEG with the addition of RNAse (3–5 μL/mL). Add TEG (Tris-EDTA-Glucose buffer) at sterile conditions, to avoid contamination. 5. Add 10 mL of a lysis solution (0.2N NaOH; 1% SDS) to the cell suspension. Gently mix by inverting the tube until all cells are lysed. It is important to mix the tube gently by inverting to avoid contamination by genomic DNA. 6. Add 7.5 mL of 3M sodium acetate (pH = 5.0) and mix thoroughly. Incubate the sample for 10 min at 4 °C. Note: If the precipitate does not settle well or flocs remain in the solution after the centrifugation, then the volume of the sodium acetate solution may be increased up to 10 mL or its pH is brought to 4.8. 7. Centrifuge the sample for 15 min at 4000 g at 4 °C. 8. Transfer the supernatant that contains the plasmid DNA to a new tube and add an equal volume of isopropanol. Thoroughly mix and incubate the sample for 15 min at room temperature. It is important to use high quality isopropanol as this directly affects the purity of the DNA. 9. Centrifuge for 20 min at 4000 g at room temperature. 10. Drain the supernatant and dry the precipitate. Dissolve the precipitate in 1 mL of sterile water. 11. Add 1 mL of 9M LiCl to the sample, mix thoroughly and incubate 20 min at - 20 °C. 12. Centrifuge for 20 min at 4000 g at - 4 °C. 13. Carefully transfer the supernatant to a new centrifuge tube (V = 15 mL). Discard the precipitate (it contains RNA and protein residues). 14. Add 5 mL of 96% ethanol to the supernatant. Vortex thoroughly. 15. Incubate for 1 h at −20 °C. PAUSE STEP It is possible to pause the protocol here and leave samples at −20 °C overnight. 16. Centrifuge at 4000 g for 20 min at 4 °C. 17. Discard the supernatant and thoroughly dry the precipitate. 18. Dissolve the plasmid DNA in 500 μL of sterile water or TE buffer.

3.2. Transfection of Human Cell Culture HEK293T Using PEI—Time for Completion: 3–5 Days 1. One day before the transfection, seed HEK293T cells into a multi-well culture plate or a tissue culture Methodsdish Protoc. with2020 respect, 3, 69 to the required number of cells per 1 cm2 (the approximate number of cells for 5 of 9 different culture dishes is shown in Table 1). Cultivate the cells overnight in DMEM supplemented with 10% FBS, 1% L-glutamine, and 1% penicillin/streptomycin. CRITICALCRITICAL STEP STEP It is Itcrucial is crucial to check to the check cells thefor mycoplasma cells for mycoplasma contamination; contamination; the presence of the presence ofmycoplasma mycoplasma may significantly may significantly reduce the reduce efficiency the of etheffi ciencytransfection of the or transduction. transfection or transduction. 2. Next day right before transfection prepare the DNA and PEI solution (transfection mix). The volume 2. Next day right before transfection prepare the DNA and PEI solution (transfection mix). of transfection mix is 10% of the total volume of the culture medium (recommended by the Themanufacturer volume of of culture transfection dishes). Add mix the is 10%required of theamount total of volumeplasmid DNA of the (see culture Table 1) medium to the Opti- (recommended byMEM the and manufacturer mix well on a ofvortex. culture Then, dishes). dropwise Add add the PEI required(1 mg/mL) amount to the DNA of plasmid solution while DNA (see Table1) tovortexing. the Opti-MEM and mix well on a vortex. Then, dropwise add PEI (1 mg/mL) to the DNA solutionNote: We recommend while vortexing. using plasmid DNA in a final concentration 1–2 mg/mL. The amount of PEI Note: We recommend using plasmid DNA in a final concentration 1–2 mg/mL. The amount of PEI may vary according to the amount of DNA. Avoid using concentrations higher than 5 µL/mL due to its high toxicity. 3. Incubate the transfection mix at room temperature for 10–15 min. 4. Remove the medium from the cells and add the required amount of fresh DMEM supplemented with 10% FBS, 1% L-glutamine, and 1% penicillin/streptomycin. Note: The presence/absence of an antibiotic in the medium does not affect the efficiency of transfection. It is possible to use Opti-MEM instead of DMEM. 5. Add the transfection mix dropwise to the cells with a fresh culture medium and mix by gently swirling the culture dish.

6. Incubate the cells with the transfection mix in a tissue culture incubator at 37 ◦C, 5% CO2 for 18–24 h. 7. After the incubation, aspirate the medium with the transfection mix from the cells and add the required amount of DMEM supplemented with 10% FBS, 1% L-glutamine, and 1% penicillin/streptomycin. 8. After 48–72 h of transfection, the effectiveness of the transfection can be evaluated. Note: For plasmids with fluorescent protein the effectiveness of the transfection can be evaluated by flow cytometry or fluorescent microscope. For other proteins use antibody or other staining.

Table 1. Approximate numbers of cells and reagents for HEK293T transfection.

Number of Cells Final Medium Volume of the Amount of Amount of Tissue Culture Plate per Well to Seed Volume (mL) Transfection Mix (µL) DNA (µg) PEI (µL) 48-well plate 40,000 0.5 50 0.5 2.5 24-well plate 80,000 1 100 1 5 12-well plate 150,000 2 200 2 10 6-well plate 300,000 3 300 3 15 60 mm culture dish 1,000,000 5 500 5 25 100 mm culture dish 3,500,000 10 1000 10 50

3.3. Lentiviral Vector Production—Time for Completion: 5–7 Days

3.3.1. Preparing Cells and Transfection 1. Seed 1 106 HEK293T cells into a 100 mm culture plate three days before the planned transfection × with lentiviral vector plasmids. On the day of transfection, cells should cover about 70% of the plate surface. Note: Take four 100 mm culture plates for production of a suﬃcient amount of vector for further research. For small-scale purposes, one well of a 6-well plate may be enough. OPTIONAL STEP We recommend to seed cells on Friday and make the transfection on Monday after the weekend so the whole process will be ended at the end of the working week. OPTIONAL STEP We recommend to coat the surface of culture plates with gelatin (ATCC, USA) or collagen (Sigma-Aldrich, USA) for better adhesion of cells during experiments. 2. Transfect cells with three plasmids necessary for the virus production: pLenti-CMV-GFP Hygro (656-4) [8], PAX2 and pMD2. Right before transfection, prepare the DNA and PEI solutions. The volume of transfection mix is 10% of the total volume of the culture medium. Add the Methods Protoc. 2020, 3, x FOR PEER REVIEW 4 of 9

3. Procedure

3.1. Plasmid DNA Isolation from E. coli Bacteria—Time for Completion: 16–18 h 1. Inoculate a single colony transformed with the desired plasmid from the plate with LB (+ antibiotic for selection) into 50 mL of liquid medium (LB or TBR) with the appropriate antibiotic added. Note: The flask for growing the bacteria should be at least 4–5 times larger than the volume of the medium in which the bacteria grow. This is necessary for a better aeration of the culture. 2. Grow the cells with vigorous shaking overnight at 37 °C. 3. Transfer the cells to centrifuge tubes (V = 50 mL) and centrifuge at 4000 g for 10 min at room temperature. 4. Carefully drain the supernatant and resuspend the cell pellet in 5 mL of sterile water or TEG with the addition of RNAse (3–5 μL/mL). Add TEG (Tris-EDTA-Glucose buffer) at sterile conditions, to avoid contamination. 5. Add 10 mL of a lysis solution (0.2N NaOH; 1% SDS) to the cell suspension. Gently mix by inverting the tube until all cells are lysed. It is important to mix the tube gently by inverting to avoid contamination by genomic DNA. 6. Add 7.5 mL of 3M sodium acetate (pH = 5.0) and mix thoroughly. Incubate the sample for 10 min at 4 °C. MethodsNote: Protoc. If the2020 precipitate, 3, 69 does not settle well or flocs remain in the solution after the centrifugation, 6 of 9 then the volume of the sodium acetate solution may be increased up to 10 mL or its pH is brought to 4.8. 7. requiredCentrifuge amount the sample of plasmidfor 15 min DNA at 4000 (see g at Table4 °C. 2) to the Opti-MEM and mix well on a vortex. Then, 8. dropwiseTransfer the add supernatant PEI (1 mg that/mL) contains to the the DNAplasmid solution DNA to a while new tube vortexing. and add an equal volume of isopropanol. Thoroughly mix and incubate the sample for 15 min at room temperature. It is important to use high quality isopropanol as this directly affects the purity of the DNA. Table 2. Approximate number of cells and reagents for virus production in HEK293T. 9. Centrifuge for 20 min at 4000 g at room temperature. 10. Drain the supernatantNumber ofand Cells dry to the precipitate.Final VolumeDissolve of the Amountprecipitate of inAmount 1 mL of sterileAmount water. of Amount of Tissue Culture Plate Seed Three Days Medium Transfection Vector PAX pMD2 11. Add 1 mL of 9M LiCl to the sample, mix thoroughly and incubate 20 min at - 20 °C. PEI (µL) before Transfection Volume (mL) Mix (µL) DNA (µg) Plasmid (µg) Plasmid (µg)

12. Centrifuge6-well plate for 20 min 100,000 at 4000 g at - 4 °C. 3 300 3.5 2.5 1.3 15 13. Carefully60 mm plate transfer the 300,000 supernatant to 6a new centri 600fuge tube (V = 6.5 15 mL). Discard 5 the precipitate 2.5 30 100(it mm contains culture dish RNA and 1,000,000 protein residues). 10 1000 11 8 4 50 14. Add 5 mL of 96% ethanol to the supernatant. Vortex thoroughly. 3.15. IncubateIncubate for the 1 h transfection at −20 °C. mix at room temperature for 10–15 min. PAUSE STEP It is possible to pause the protocol here and leave samples at −20 °C overnight. 4. Change the media containing the transfection mix to fresh DMEM supplemented with 10% FBS, 16. Centrifuge at 4000 g for 20 min at 4 °C. 17. 1%Discard L-glutamine, the supernatant and and 1% penicillinthoroughly/ streptomycin.dry the precipitate. 5.18. AddDissolve the the transfection plasmid DNA mix in 500 dropwise μL of sterile to the water cells or withTE buffer. a fresh culture medium and mix by gently swirling the culture dish. 3.2. Transfection of Human Cell Culture HEK293T Using PEI—Time for Completion: 3–5 Days 6. Incubate the cells with the transfection mix in a tissue culture incubator at 37 ◦C, 5% CO2 for 1. 18–24One day h. before the transfection, seed HEK293T cells into a multi-well culture plate or a tissue culture dish with respect to the required number of cells per 1 cm2 (the approximate number of cells for 7. The next day after transfection, change the medium to fresh DMEM supplemented with 10% FBS, different culture dishes is shown in Table 1). Cultivate the cells overnight in DMEM supplemented 1%with L-glutamine, 10% FBS, and 1%1% penicillin L-glutamine,/streptomycin. and 1% penicillin/streptomycin. CRITICALCRITICAL STEP STEP It is crucialThis to and check all the following cells for mycoplasma steps must contamination; be done withthe presence the proper of biosafety containmentmycoplasma may recommended significantly reduce for researchthe efficiency with of the lentiviral transfection vectors or transduction. [11]. 2. Next day right before transfection prepare the DNA and PEI solution (transfection mix). The volume 3.3.2.of Harvesting transfection themix Lentiviral is 10% of Vectorsthe total volume of the culture medium (recommended by the manufacturer of culture dishes). Add the required amount of plasmid DNA (see Table 1) to the Opti- 8. TheMEM next and threemix well days, on a collectvortex. Then, daily dropwise the medium add PEI containing (1 mg/mL) theto the virus DNA in solution sterile while 50-mL centrifuge tubes.vortexing. Store the medium at +4 ◦C for up to one week. 9. OnNote: the We fifthrecommend day after using transfection, plasmid DNA in the a fina collectedl concentration medium 1–2 mg/mL. must The be filteredamount of with PEI a filter with a 0.45 µm filter to get rid of cell debris in the medium. OPTIONAL STEP For an easier filtration, it is possible to preliminarily centrifuge the collected medium (3 min at 500 g) to precipitate the main cell debris. × 3.3.3. Ultracentrifugation 10. Transfer the filtered virus-containing medium to sterile polycarbonate centrifuge bottles suitable for ultracentrifugation and balance them carefully using suitable weights (to 3 decimal points). CRITICAL STEP the 50 mL centrifuge tubes must be strong enough to withstand a centrifugation speed of 50,000 g. × 11. Centrifuge the tubes for 2 h at 47,000 g, at +4 C. Note: After the centrifugation, at the bottom of × ◦ the tube will be a barely noticeable whitish precipitate. 12. Take away the supernatant without touching the precipitate. 13. Thoroughly dissolve the precipitate in 150 µL of Opti-MEM per 50 mL of harvested vector- containing medium by pipetting. 14. Leave them for 1 h at room temperature for better dissolution. 15. Centrifuge samples at low speed (2000 g, 3 min) to collect the media with the virus particles. × 16. Aliquot samples into 20–50 µL portions and freeze them at 80 C. In this state, the samples can − ◦ be stored for a very long time. CRITICAL STEP We strongly recommend neither to repeat the freezing/thawing cycles for the virus nor to store it for a long time at +4 ◦C and at room temperature, since its transduction efficiency can be significantly reduced. Methods Protoc. 2020, 3, 69 7 of 9

3.3.4. Viral Titer Calculation 17. Seed 5 104 HEK293T cells into a 24-well plate the day before transduction. Cultivate the cells × overnight in DMEM supplemented with 10% FBS, 1% L-glutamine, and 1% penicillin/streptomycin. 18. The next day, prepare the 10-fold serial dilution of your lentivirus in 500 µL of Opti-MEM. OPTIONAL STEP To increase the eﬃciency of the transduction, use hexadimethrine bromide (polybrene) (Sigma-Aldrich, USA) in concentration 5 µg/mL. This step is essential for the primary lines. 19. Change the culture medium in the culture plate to the Opti-MEM medium with diﬀerent dilutions of the virus (see Point 18) to infect the cells.

20. Incubate the cells in a tissue culture incubator at 37 ◦C, 5% CO2 for 18–24 h. 21. The next day after transduction, change the medium to fresh DMEM supplemented with 10% FBS, 1% L-glutamine, and 1% penicillin/streptomycin. 22. Two days after transduction, perform a flow cytometry analysis to count the percentage of the infected cells per well. To detect the infected cells, use the fluorescence signal value for viruses containing a fluorescent protein or antibody/other staining for non-fluorescent ones. 23. Choose the well in which the percentage of infected cells do not exceed 20% and calculate the viral titer using the following equation: TU/mL = (P N D)/V, where TU/mL is a number of × × viral particles in 1 mL of viral solution; P—percentage of infected cells (0.01–0.2); N—the number of cells in the well (at the time of seeding); D—the dilution fold of the added vector; and V—the volume (mL) of the added vector.

4. Expected Results During the approbation of the protocol for the isolation of plasmid DNA, we received on average of 5.6 2.1 µg/µL (which is about 56 µg of DNA per 1 mL of overnight culture). We obtained such ± amounts of plasmid DNA using the bacterial strain STBL3. The purity of the plasmids and the concentration was evaluated using the spectrophotometer NanoDrop (ND-1000). This is a quick method for estimating the amount of extracted DNA, as well as the relative amount of RNA and protein in a sample. The ratio of absorbance at 260 nm and 230 nm (260/230) is used as a secondary measure of nucleic acid purity. The 260/230 values for pure nucleic acid are often higher than the respective 260/280 values. A good-quality DNA sample should have an absorbance 260/280 ratio of 1.8–2.0 and a 260/230 ratio of 2.0–2.2 [12]. The average absorbance at 260/280 nm for the samples obtained by this method was 1.94 0.09 and the average absorbance at 260/230 nm was 2.23 0.2. ± ± Plasmids extracted with the described method were successfully used for transfection of HEK293T cells and lentivirus production. Transfection of mammalian cell cultures is a very powerful tool in various biological fields. We used a basic protocol using the cationic polymer PEI. This method is suitable for different purposes and can be done with good efficiency in different stable cell lines. The efficiency of HEK293T transfection on the third day was more than 90% (measured using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA)). The protocol for the production of lentiviral vectors is suitable for various lentiviral transfer plasmids with an insert size up to 15 kb [13]. Using this method for the production of lentiviruses, using the plasmids pMD2 (Addgene plasmid #12259), PAX2 (Addgene plasmid #12260) and pLenti-CMV-GFP Hygro (656-4) [8], we obtained viral titers up to 1.5 108 TU/mL. Measurements of the calculation of the efficiency of transduction × with lentivirus were carried out on a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA). As an example, we present a photo of HEK293T for three days during the virus production (Figure2). Figure2 compares two techniques: the bright field presents a number of cells infected with the virus Methods Protoc. 2020, 3, x FOR PEER REVIEW 8 of 9

obtained viral titers up to 1.5 × 108 TU/mL. Measurements of the calculation of the efficiency of transduction with lentivirus were carried out on a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA). As an example, we present a photo of HEK293T for three days during the virus production (Figure 2). Figure 2 compares two techniques: the bright field presents a number of cells infected with Methods Protoc. 2020, 3, 69 8 of 9 the virus and their morphological alterations, while GFP depicts the viral assembly. With the three days of growth, an increase in the GFP intensity is noticeable and so too the assembled viral particles. We describe the obtaining of LV-CMV-EGFP Hygro (656-4) that has a good transduction and their morphological alterations, while GFP depicts the viral assembly. With the three days of efficiency with both immortalized (more than 98% efficiency on the third day) and primary growth, an increase in the GFP intensity is noticeable and so too the assembled viral particles. mammalian cell lines (60–80% efficiency on the third day, depending on the cell type).

FigureFigure 2. Dynamics 2. Dynamics of GFP of GFP signal signal accumulation accumulation in in HEK293T HEK293T within within three three days days duringduring thethe LV-CMV-EGFPLV-CMV- EGFP Hygro (656-4) virus production. BF, brightfield; GFP, detection of green fluorescence. HEK293T Hygro (656-4) virus production. BF, brightfield; GFP, detection of green fluorescence. HEK293T cells cells 24 hours (A,D), 48 hours (B,E) and 72 hours (С,F) after initial transfection with plasmids for LV- 24 h (A,D), 48 h (B,E) and 72 h (C,F) after initial transfection with plasmids for LV-CMV-EGFP Hygro CMV-EGFP Hygro (656-4) production. (656-4) production. 5. Reagents Setup We describe the obtaining of LV-CMV-EGFP Hygro (656-4) that has a good transduction efficiency with bothLB immortalized. 1% tryptone, (more 0.5% yeast than extract, 98% effi andciency 1% NaCl. on the To third obtain day) a solid and medium, primary add mammalian agar to a final cell lines concentration of 2%. Add deionized water. Shake until the solutes are dissolved and autoclave for (60–80% efficiency on the third day, depending on the cell type). 30 min. Store liquid medium at room temperature (or at + 4 °C) and add the appropriate antibiotics 5. Reagentsbefore use. Setup In the solid medium, add antibiotics after cooling the medium to 45–50 °C and pour into Petri dishes. Store at 4 °C. LB. 1%TBR. tryptone, Deionized 0.5% water yeast up to extract, 900 mL, and 12 g 1% tryptone, NaCl. 24 To g obtain yeast extract, a solid and medium, 4 mL glycerol. add agar Shake to a final concentrationuntil the solutes of 2%. are Add dissolved deionized and autoclave water. Shake for 30 until min. theAdd solutes 100 mL are of sterile dissolved solution and of autoclave 0.17 M for 30 min.KH2 StorePO4, 0.72 liquid M K medium2HPO4 (2.3 at g roomof K2HPO temperature4 and 12.5 g (or of atKH+24PO◦C)4 in and 90 mL add ofthe H2O; appropriate after the salts antibiotics are dissolved, add H2O up to 100 mL). before use. In the solid medium, add antibiotics after cooling the medium to 45–50 ◦C and pour into TEG. 50 mM Glucose, 25 mM Tris HCl (pH = 8.0), and 10 mM EDTA (pH = 8.0). Add deionized Petri dishes. Store at 4 ◦C. water. Autoclave for 30 min. Can be stored at room temperature. TBR. Deionized water up to 900 mL, 12 g tryptone, 24 g yeast extract, and 4 mL glycerol. Shake Lysis solution. 0.2 N NaOH (freshly diluted from a 5 N stock) and 1% (w/v) SDS (freshly diluted untilfrom the solutesa 10% stock). are dissolved Store solution and at autoclave room temperature. for 30 min. Add 100 mL of sterile solution of 0.17 M KH2PO4,10× 0.72 TE M buffer. K2HPO 1004 (2.3mM gTris-HCl of K2HPO (pH4 =and 8.0), 12.5 10 mM g of EDTA. KH2PO Sterilize4 in 90 solution mL of Hby2O; autoclaving after the saltsfor are dissolved,30 min. add Store H the2O buffer up to at 100 room mL). temperature. TEG.Polyethylenimine.50 mM Glucose, For 25 the mM PEI Tris (1 HClmg/mL) (pH preparation,= 8.0), and se 10e the mM recommendations EDTA (pH = 8.0). written Add by deionized the water.manufacturer Autoclave [14]. for 30 min. Can be stored at room temperature. Lysis solution. 0.2 N NaOH (freshly diluted from a 5 N stock) and 1% (w/v) SDS (freshly diluted from a 10% stock). Store solution at room temperature. 10 TE buffer. 100 mM Tris-HCl (pH = 8.0), 10 mM EDTA. Sterilize solution by autoclaving for × 30 min. Store the buffer at room temperature. Polyethylenimine. For the PEI (1 mg/mL) preparation, see the recommendations written by the manufacturer [14].

Author Contributions: Conceptualization, D.V.K. and J.I.K; validation, D.V.K., J.I.K., J.S.I. and A.A.R.; writing— original draft preparation, D.V.K.; writing—review and editing, D.V.K., J.I.K., J.S.I. and A.A.R.; visualization D.V.K. Methods Protoc. 2020, 3, 69 9 of 9

and J.I.K.; supervision, A.A.R.; methodology, D.V.K. and J.I.K.; funding acquisition, A.A.R. All authors have read and agreed to the published version of the manuscript. Funding: The reported study was funded by a Russian Science Foundation grant № 20-14-00148 (Lentivirus production and cell cultures transfection) (D.V.K.and A.A.R.), the Russian Foundation of Basic Research 19-34-90153 (D.V.K.) and 19-34-90146 (J.I.K.), and by project 51140332 from the St. Petersburg State University (D.V.K. and A.A.R.). Acknowledgments: The authors are grateful to Julia V. Sopova (Laboratory of Amyloid Biology, St. Petersburg State University) for valuable advice and comments while working on the protocol and Maria S. Rubel (SCAMT Intitute, ITMO University) for guidance and valuable comments in preparing the publication. We also acknowledge the St. Petersburg State University Resource Centers «Molecular and Cell Technologies», «CHROMAS» and «Biobank» for technical support. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

1. Wurm, F.M. Production of recombinant protein therapeutics in cultivated mammalian cells. Nat. Biotechnol. 2004, 22, 1393–1398. [CrossRef][PubMed] 2. Kim, T.K.; Eberwine, J.H. Mammalian cell transfection: The present and the future. Anal. Bioanal. Chem. 2010, 397, 3173–3178. [CrossRef][PubMed] 3. Benskey, M.J.; Manfredsson, F.P. Lentivirus Production and Purification. Methods Mol. Biol. 2016, 1382, 107–114. [CrossRef] [PubMed] 4. Longo, P.A.; Kavran, J.M.; Kim, M.S.; Leahy, D.J. Transient mammalian cell transfection with polyethylenimine (PEI). Meth. Enzymol. 2013, 529, 227–240. [CrossRef][PubMed] 5. Sakuma, T.; Barry, M.A.; Ikeda, Y. Lentiviral vectors: Basic to translational. Biochem. J. 2012, 443, 603–618. [CrossRef][PubMed] 6. Jiang, W.; Hua, R.; Wei, M.; Li, C.; Qiu, Z.; Yang, X.; Zhang, C. An optimized method for high-titer lentivirus preparations without ultracentrifugation. Sci. Rep. 2015, 5, 13875. [CrossRef][PubMed] 7. Inoue, H.; Nojima, H.; Okayama, H. High eﬃciency transformation of Escherichia coli with plasmids. Gene 1990, 96, 23–28. [CrossRef] 8. Campeau, E.; Ruhl, V.E.; Rodier, F.; Smith, C.L.; Rahmberg, B.L.; Fuss, J.O.; Campisi, J.; Yaswen, P.; Cooper, P.K.; Kaufman, P.D. A versatile viral system for expression and depletion of proteins in mammalian cells. PLoS ONE 2009, 4, e6529. [CrossRef] [PubMed] 9. Birnboim, H.C.; Doly, J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979, 7, 1513–1523. [CrossRef][PubMed] 10. Birnboim, H.C. A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol. 1983, 100, 243–255. [CrossRef][PubMed] 11. Biosafety Considerations for Research with Lentiviral Vectors. Available online: https://osp.od.nih.gov/wp- content/uploads/Lenti_Containment_Guidance.pdf (accessed on 14 October 2020). 12. Desjardins, P.; Conklin, D. NanoDrop Microvolume Quantitation of Nucleic Acids. JoVE 2010, 2565. [CrossRef] [PubMed] 13. Counsell, J.R.; Asgarian, Z.; Meng, J.; Ferrer, V.; Vink, C.A.; Howe, S.J.; Waddington, S.N.; Thrasher, A.J.; Muntoni, F.; Morgan, J.E.; et al. Lentiviral vectors can be used for full-length dystrophin gene therapy. Sci. Rep. 2017, 7, 79. [CrossRef] [PubMed] 14. Polysciences. Available online: https://www.polysciences.com/skin/frontend/default/polysciences/pdf/24765_ usage_guide.pdf (accessed on 28 August 2020).

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