Chapter 8 Lentiviral Transduction of Mammary Epithelial Cells Richard Iggo and Elodie Richard Abstract Lentiviral vectors are the workhorses of modern cell biology. They can infect a wide variety of cells including nondividing cells and stem cells. They integrate into the genome of infected cells leading to stable expression. It is easy to transduce 100 % of the cells in a culture and possible to infect cells simultaneously with multiple vectors, greatly facilitating studies on malignant transformation. We present simple protocols to produce and titrate lentiviral vectors, infect mammary epithelial cells, and check for contamination with replication-competent viruses. Key words Lentivirus, Transfection, Infection
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Introduction Lentiviral vectors are convenient tools to express transgenes in a wide variety of cultured cells. At first glance it might seem somewhat risky to use vectors derived from HIV as basic research tools, but the labs that conceived them were aware of the risks and went to great lengths to make them safe [1–4]. Additional information on safety is given in Notes 1 and 2. Minimally, you should familiarize yourself with level 2 biosafety precautions and make the requisite declarations to the authorities before starting to work with lentiviral vectors. Unlike transgenes in transfected DNA, transgenes in lentiviral vectors are immediately integrated stably into the genome of the target cell, greatly increasing the chance that they will be expressed stably. Unlike simple oncoretroviral or gamma retroviral vectors, lentiviral vectors can infect nondividing cells. The critical difference is that the cDNA produced by reverse transcription of lentiviral RNA is professionally imported into the nucleus, whereas cDNA from simple retroviruses must wait for the nuclear envelope to break down at mitosis to gain access to the chromosomes. This simplifies the protocol because it is not necessary to passage the target cells immediately after infection to make them divide.
Maria del Mar Vivanco (ed.), Mammary Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 1293, DOI 10.1007/978-1-4939-2519-3_8, © Springer Science+Business Media New York 2015
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A second advantage is that titers tend to be higher and virus can be frozen with minimal loss of titer. The vectors are normally pseudotyped with vesicular stomatitis glycoprotein (VSV G), which broadens the tropism because it works by inducing fusion with the plasma membrane. This works well for mammary epithelial cells, which are normally easy to infect with VSV G pseudotyped lentiviral vectors.
2
Overall Strategy The basic approach to make lentiviral vectors is to transfect 293T cells with a vector plasmid and two or three helper plasmids. The protocol below uses calcium phosphate transfection because it is cheap and highly effective in 293T cells. The transfection efficiency should be close to 100 %; if you find it difficult to achieve this, try commercial transfection reagents instead, such as Fugene (Promega).
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Packaging Vectors Most labs use so-called second-generation plasmids, in which one helper plasmid contains all the lentiviral helper genes and a second helper plasmid contains VSV G. Other than tat and rev, the lentiviral accessory genes (vif, vpr, vpu, and nef) are all deleted from the helper plasmids. In third-generation systems the lentiviral genes are split between two plasmids, with the gag, polymerase, protease, and integrase genes on one plasmid and rev on another. In this configuration the tat gene is deleted, which means the vector plasmid requires a tat-independent promoter. A plasmid expressing VSV G is still required, meaning a total of four plasmids must be transfected. The third-generation system is considered safer but the need to transfect an additional plasmid may reduce the titer. In practice, most users choose the second-generation psPAX2 and pMD2.G packaging vectors developed by the Naldini and Trono labs (Fig. 1a, b, Addgene 12259 and 12260).
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Lentiviral Expression Vectors There is a much wider choice of vector plasmids. In addition to deletion of all complete HIV genes, current vectors have partial replacement of the 5′-LTR by a heterologous promoter to make transcription in the packaging cell independent of tat; a deletion in the 3′-LTR to make both LTRs inactive as promoters after integration; and the presence of an RNase H-resistant central polypurine tract that primes reverse transcription and promotes nuclear import of
CMV enhancer Chicken beta actin promoter
ampR (bla)
actin intron
ColE1 origin SV40 ori psPAX2
HIV Gag
10703 bp
beta-globin polyA
HIV Protease HIV Tat/Rev HIV RRE HIV Polymerase HIV cPPT HIV Integrase
CMV promoter
ampR (bla)
beta globin intron pMD2.G pBR322 ORI
5824 bp
VSV-G beta globin polyA
Fig. 1 Lentiviral plasmids. (a) psPAX2 contains the main lentiviral genes required for packaging: gag, protease, polymerase, integrase, tat, and rev. (b) pMD2.G expresses VSV glycoprotein, which replaces the normal envelope protein. VSV G fuses directly with the plasma membrane, allowing the viral particles to infect cells indiscriminately. (c) pSD-82 is a typical lentiviral expression vector. The genomic transcripts made in the packaging cells initiate at a hybrid RSV/HIV promoter. The RSV enhancer is deleted in integrated proviruses because the 3′-LTR is copied to the 5′-LTR during reverse transcription. There is a deletion removing the enhancer in the U3 region in the 3′-LTR. This deletion is copied to the 5′-LTR after reverse transcription to create integrated proviruses with defective promoters in both LTRs (a so-called self-inactivating or SIN vector). This means the first transcripts from the integrated proviruses initiate at the human PGK promoter. Since this is located after the packaging signal (psi), no transcripts from the integrated provirus can be packaged. The transgene, ESR1, is located between two Gateway attB sites and expressed from the human PGK promoter. The WPRE located after the attB2 site increases the level of the ESR1 RNA. The puromycin resistance gene is expressed from the murine PGK promoter. The viral RNAs all terminate in the 3′-LTR. It is important to delete polyA signals from the transgene to avoid cleaving the 3′-end off the genomic RNA that is packaged in the viral particles
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pUC ORI
RSV LTR U3 HIV LTR RU5 psi RRE
ampR cPPT pSD-82 F1 ORI
hPGK promoter
9676 bp attB1
SV40 ORI HIV SIN LTR U3'RU5 puroR (pac) mPGK promoter
ESR1 orf attB2 WPRE
Fig. 1 (continued)
the preintegration complex. Many companies now sell lentiviral vectors but a cheap and convenient option for academic labs is to use vectors from Addgene. In addition to specific vectors that have been described in publications, Addgene contains an impressive collection of vectors deposited by Eric Campeau, many of which are based on the Gateway cloning system (http://www.addgene. org/Eric_Campeau/). The Campeau vectors systematically offer antibiotic resistance cassettes for puromycin, neomycin, hygromycin, blastomycin, and zeomycin, making it possible to select for multiple different vectors in the same cell. We have had difficulty selecting for hygromycin in primary mammary epithelial cells but the other markers all work well. The choice of promoter is dictated by the propensity of some promoters to become silenced more easily than others (cellular promoters are often better than viral promoters), and by the characteristics of the cells transduced (PGK works well in mammary progenitors and stem cells). The vector we have used most extensively in mammary epithelial cells has a human PGK promoter driving the transgene and a mouse PGK promoter driving the puromycin resistance gene (pac) (Fig. 1c) [5]. Note that the vector contains a Woodchuck hepatitis virus RNA stability element (WPRE) between the two expression cassettes, so the WPRE is only present in the RNA produced by the promoter driving expression of the transgene. This is a good idea in the sense that it increases the chance of the transgene being expressed well, but careful titration of puromycin is required to avoid killing the cells if they are infected at single copy. Many of the Campeau vectors have the same promoters in the same configuration and express transgenes well in mammary epithelial cells.
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Materials
5.1 Components for Lentiviral Production
293T/17 cells (ATCC CRL-11268). Dulbecco’s modified Eagle medium, 10 % FBS. Phosphate-buffered saline. 0.05 % trypsin/EDTA. TE buffer, pH 8.0. DNase/RNase-free water. Polylysine. 2.5 M CaCl2, 18.4 g for 50 ml; filter sterilize (0.22 μm) and store aliquots at −20 °C. 2 M HEPES, 52.6 g for 100 ml; filter sterilize (0.22 μm) and store aliquots at −20 °C. OptiMEM (Life Technologies); add 2 M HEPES to make OptiMEM 20 mM HEPES. HEPES-buffered saline (2× HeBS, Sigma 51558—50 ml). 10 cm tissue culture dishes. 15 and 50 ml conical base tubes. 0.45 and 0.22 μm syringe filters. Vivaspin 20 MWCO 100,000 (GE Healthcare #28-9323-63). 5 % CO2, 37 °C incubator. 1 % Virkon (50 g sachets, Dupont). 70 % (v/v) ethanol. Plasmids to make a simple GFP expression vector pRRLSIN.cPPT.PGK-GFP.WPRE (classic GFP expression vector, Addgene 12252). pMD2.G (for pseudotyping with VSV G protein, Addgene 12259). psPAX2 (contains the main HIV-1 helper genes: Gag, Pol, Tat, and Rev, Addgene 12260). Many other useful plasmids can be obtained from Addgene. See Notes 3 and 4 for information on construction and production of plasmids. All plasmid maxipreps should be “endotoxin-free” (Qiagen EndoFree or Nucleobond Xtra EF). For convenience, adjust the concentration of plasmids to 1 mg/ml.
5.2 Components for Titration of Viruses
Target cells (e.g., MCF7 cells). Cell culture medium (RPMI 10 % FCS for MCF7 cells). Viral suspension. Phosphate-buffered saline.
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0.05 % trypsin/EDTA. 6-Well plates. FACS tubes (polypropylene). 5 % CO2, 37 °C incubator. 5.3 Components for Isolation of Mammary Epithelial Cells
Cold DMEM/F12, phenol red-free, 2× Pen/Strep = minimal medium (MM). Phosphate-buffered saline. Collagenase A: Make 100 mg/ml in PBS, and store aliquots at −20 °C. Trypsin solution 0.25 %. Dispase: Make 5 mg/ml in PBS, filter sterilize (0.22 μm), and store aliquots at −20 °C. Deoxyribonuclease 1 (DNase): Make 1 mg/ml in PBS, filter sterilize (0.22 μm), and store aliquots at −20 °C. Cell strainer 40 μm. Sterile forceps. Sterile scalpels. Sterile chopping board. 10 cm cell culture dishes. 15 and 50 ml conical base tubes.
5.4 Components for Lentiviral Transduction of Mammary Cells
Single cells from reduction mammoplasty. Cell culture medium (according to individual lab preference). Viral stock. Phosphate-buffered saline. 0.05 % trypsin/EDTA. 6-Well plates. 5 % CO2, 37 °C incubator. Use 5 % O2 for primary cultures.
5.5 Components for RCL Test
Viral suspension. HIV genomic RNA (if available). 293T/17 cells (ATCC CRL-11268). Dulbecco’s modified Eagle medium, 10 % FBS. Phosphate-buffered saline. 0.05 % trypsin/EDTA. Trizol LS. RNeasy kit. 6-Well plates. 5 % CO2, 37 °C incubator.
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Reagents for routine molecular biology Superase inhibitor, 20 U/μl. DNase I (RNase-free) 10 U/μl. Restriction buffer M (Roche 11417983001). DNase/RNase-free water. 10 % SDS. Phenol/chloroform/isoamyl alcohol. NaOAc 3 M pH 5.5. Glycogen 20 mg/ml. Ethanol 70 and 100 % (molecular biology grade). Oligo-dT 0.5 mg/ml. dNTPs (10 mM each). Superscript II (reverse transcriptase, first-strand buffer, DTT). Sybr Green qPCR master mix 2× (Life Technologies 4344463). Forward primer PAXpolF: tggcagaaaacagggagatt. Reverse primer PAXpolR: tgcccctgcttctgtatttc. qPCR machine.
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Methods Follow national biosafety level 2 guidelines when working with lentiviral vectors. If you are unsure what to do you are almost certainly not legal, so seek advice on local regulations from more experienced colleagues before starting the project. See Note 1 for additional information on biosafety of lentiviral vectors.
6.1 Production of Lentiviral Particles
293T cells grow rapidly and detach easily. Coating the dishes with polylysine helps to prevent the cells falling off the dish during medium changes. The protocol below differs from some classic protocols in that cells are plated 3 days before transfection. This is designed for a plate on Friday–transfect on Monday–harvest on Wednesday strategy. Despite the relatively high density at the time of transfection, the yields are excellent. 293T cells should be maintained in DMEM and 10 % FCS and split 1:10 twice a week. Take care always to passage the stock plates before the medium is exhausted. Replace the cultures at least every 3 months from frozen stocks that have been checked for mycoplasma. If none of the vectors contains GFP it is helpful to set up an additional transfection with GFP to check the efficiency of transfection. 1. Coat tissue culture plates with polylysine by briefly pipetting 5 ml of polylysine onto 10 cm plates.
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2. Plate 2 × 106 293T cells per 10 cm dish 3 days before transfection. 3. On the day of transfection, replace the medium with 5 ml DMEM and 10 % FCS. Cells should be about 80 % confluent when transfected (see Note 7). 4. For each 10 cm plate, prepare the following plasmid cocktails in 1.5 ml Eppendorf tubes: 4 μg pMD2.G (VSV-G viral envelope) 10 μg psPAX2 (packaging construct) 10–20 μg of your expression vector, adjusted according to the size of the plasmid (10 μg 10,000 bp) Add sterile water to 450 μl. 5. Add 50 μl CaCl2 to the plasmid cocktails and mix thoroughly. Prepare 15 ml Falcon tubes containing 500 μl 2× HeBS. 6. Add the plasmid cocktails drop by drop to the Falcon tubes containing the 2× HeBS while shaking them on a vortexer at low speed. 7. Wait for 5 min to allow the precipitate to form. 8. Add the precipitate to the cells by pipetting it drop by drop, trying to homogeneously cover the whole plate. Ensure even spreading of the precipitate by gently swirling the dish. Return the dish to the 37 °C 5 % CO2 incubator. 9. 6–14 h after transfection, remove the medium and replace it with 8 ml OptiMEM 20 mM HEPES. 10. 38–46 h after starting the transfection, harvest the medium, which now contains lentiviral particles. Centrifuge at 2,500 rpm for 3 min to remove debris, and then filter with a 0.22 μm syringe filter. The titer will drop if the medium becomes very acid, so it is not worth waiting an extra day before harvesting the virus in the hope that the yield will go up. 11. Optional: To concentrate the virus 10- to 100-fold, use a Vivaspin column (see Note 7). This is only possible if you used serum-free medium (OptiMEM) as described above (if you use FCS the filter will concentrate unwanted proteins from the serum and become clogged). Before you embark on extensive centrifugation of virus, check that you have taken reasonable steps to contain aerosols that might be released by leakage of viral supernatant. 12. Aliquot virus in freezing vials and store at −80 °C. 6.2 Titration of Lentiviral Vectors
Cells differ in their infectability, so viral titers are only strictly valid on the cell line used to calculate the titer. Since it is often not possible to perform a titration on the cells you are most interested
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in, you need to bear in mind that there may be a systematic error when you do the experiment (the effective multiplicity of infection [moi] may be higher or lower than you intended). See Note 5 for more information on calculating viral titers. 6.2.1 Titration by Antibiotic Selection
This is the simplest approach. It only gives a rough idea of the titer but this is good enough for most experiments. A dilution series is performed and the titer is based on finding the first well with no dead cells after antibiotic treatment. This well is conventionally taken to have been infected at an moi of 1. This approach works best for antibiotics that kill fairly quickly, in particular puromycin, blastomycin, and hygromycin. 1. Plate cells at a relatively low density to ensure that they can be left for a week without becoming overgrown. For MCF7 use 1 × 105 cells per well in 6-well plates, one plate per virus to be titered. 2. Infect cells. The following day, prepare six Eppendorf tubes each with a final volume of 1 ml medium containing: A. 1 μl virus B. 10 μl virus C. 100 μl virus D. 100 μl virus (control for toxicity of virus) E. No virus (control for toxicity of virus) F. No virus (control for antibiotic efficacy) 3. Replace the medium on the cells with the 1 ml dilutions in the Eppendorfs. 4. Incubate for 6 h, then remove the medium, and add 2 ml of fresh medium. 5. Antibiotic selection: 48 h after infection, remove medium and add 2 ml of fresh medium containing antibiotic to wells A, B, C, and F. Add 2 ml of fresh medium without antibiotic to wells D and E. The concentration of antibiotic required depends on the cells, so before testing the virus you should titrate the antibiotic to find the concentration that kills uninfected cells in a reasonable time—for MCF7 it is likely to be around 2 μg/ml puromycin, 200 μg/ml hygromycin, 5 μg/ml blasticidin, 750 μg/ml G418, and 1 mg/ml zeomycin. The time to kill 100 % of the cells should be around 3 days for puromycin, 5 days for blasticidin and hygromycin, 10 days for zeomycin, and 14 days for G418. 6. Calculate viral titer. Observe the cells and score them when there is obvious death in well F. This will be 2–3 days for puromycin, and up to 1 week for the other antibiotics. G418 is so slow that it may be hard to interpret the results because the
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cells are too confluent. The goal is to find the well with the lowest amount of virus giving no death. Compare wells D and E to check that death is due to antibiotic rather than a direct toxic effect of the virus. Titer (infectious particles/ml) = number of cells at infection/ volume of virus in ml in the first well with no death. If you used the amounts above, the titers will be: 1 × 105/0.001 = 1 × 108 infectious particles/ml if there is no death in well A 1 × 105/0.01 = 1 × 107 infectious particles/ml if there is no death in well B 1 × 105/0.1 = 1 × 106 infectious particles/ml if there is no death in well C Good titers are 107–108 infectious particles/ml in unconcentrated viral supernatant. This approach will slightly underestimate the titer (see Note 5 for the reason why). 6.2.2 Titration by Flow Cytometry
It is possible to titer viruses accurately by flow cytometry provided they express a fluorescent protein such as GFP. It is the percentage of cells positive not the intensity of expression that matters. 1. Plate 1 × 105 MCF7 cells per well in 6-well plates, one plate per virus to be tested. Seed two additional wells that will not be infected, so you can use one to count the exact number of cells at the time of infection and the other as a negative control to set the flow cytometer gates. 2. Infect cells. The day after plating the cells, prepare Eppendorf tubes containing serial twofold dilutions of virus. In the first tube mix 64 μl virus with 1,934 μl medium. Then set up five tubes containing 1 ml medium and make serial dilutions by transferring 1 ml sequentially to each tube. 3. Remove the medium from the cells and add 1 ml of each virus dilution per well. Harvest one uninfected well to count the number of cells at the moment of infection (this lets you correct for the plating efficiency and for cells that have divided since they were plated). 4. Incubate for 6 h, then remove the medium, and add 2 ml of fresh medium. 5. Three days after infection, aspirate the medium, add 0.5 ml trypsin/EDTA to each well, including the control uninfected well, and incubate at 37 °C until the cells detach. 6. Add 1 ml medium to neutralize the trypsin, pipette up and down to ensure that the cells are dissociated, and then transfer the suspension to 5 ml polypropylene FACS tubes.
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7. Pellet the cells by centrifuging for 5 min at 1,500 rpm and discard the supernatant. Optional: If your flow cytometry facility does not accept cells recently transduced with level 2 viruses, kill residual virus by resuspending the pellet in 0.5 % formaldehyde in PBS and incubating at room temperature for 5 min. Then pellet the cells again by centrifuging for 5 min at 1,500 rpm and discard the supernatant. Note that formaldehyde is compatible with GFP fluorescence but may abolish the fluorescence of other markers. 8. Resuspend the cells in 200 μl of PBS, store them at 4 °C, and perform flow cytometry within a few hours. Use the uninfected control well to set the gate for background fluorescence. 9. Record the percentage of infected cells in each well. 10. In Excel, make a table with the volume of virus in ml in one column and the percentage of fluorescent cells in another column. Add a column called “moi” in which you enter a formula for the “negative log of the fraction of cells not infected.” The formula should look like this: = -LN ( (100 - POS ) / 100 ) where POS refers back to the column with the percentage of fluorescent cells, and LN() takes the natural log. The “moi” column now contains the moi achieved with the specified amount of virus. 11. Make a graph with volume of virus in ml on the x-axis and “moi” on the y-axis. Exclude any obvious outliers (they may occur at the most extreme virus concentrations), then invite Excel to draw a straight line through the points, and ask it to display the slope and intercept of the line. The viral titer in infectious units per ml is given by titer = ( slope ´ cell number ) / (1 - intercept ) where cell number is the number of cells present in the well at the time of infection. See Note 5 for additional information on calculation of titers. 6.2.3 Titration by p24 ELISA
It is possible to titer lentiviral vectors by ELISA for p24, a capsid protein derived from the gag polyprotein. This approach measures the amount of p24 in pg, which depends on the number of physical virus particles. The ratio of infectious units to physical particles (typically 1 in 100 to 1 in 1,000) is used to assess the quality of viruses, for example to detect batch-to-batch variation in the quality of preps destined for clinical use. Since the quality of typical
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academic lab virus preps is highly questionable, it is necessary to make some very large assumptions when extrapolating from physical titers to infectious titers. The approach is only useful if you have a burning desire to know the titer and there is no other way to do it (no antibiotic marker, no fluorescent marker, no transgene that can be detected in transduced cells by IF or IHC). When forced to measure physical titers we use the Innotest HIV Antigen mAb P24 ELISA kit (#80563, http://www.fujirebio-europe.com) according to the manufacturer’s instructions. 6.3 Isolation of Mammary Epithelial Cells
The protocol can conveniently be split into two parts separated by the freezing of the organoids. 1. Place reduction mammoplasty tissue on chopping board. 2. Remove yellow fatty tissue by scraping it off with a scalpel. 3. Shear and chop the remaining tissue using two scalpels until it reaches a mushy consistency. 4. Add 1 g tissue to 10 ml MM + 100 μl Collagenase in 50 ml tube. 5. Incubate at 37 °C with agitation (roller) for 3–16 h. 6. Spin at 1,400 rpm (~400 × g) for 4 min. 7. Wash pellet twice with warm PBS and 2 % FCS. 8. Resuspend pellet (“organoids”) in 2 ml freezing medium (10 % DMSO, 90 % FBS; or Cryo3). 9. Aliquot into cryotubes and freeze in isopropanol box (Mr. Frosty, Nalgene) at −80 °C overnight and then transfer to liquid nitrogen. 10. Thaw organoids and transfer to 15 ml tube containing 4 ml prewarmed MM medium. 11. Spin at 1,400 rpm (~400 × g) for 1 min. 12. Eliminate the supernatant carefully. 13. Add 500 μl prewarmed trypsin 0.25 % and resuspend organoids. 14. Incubate for 2 min at room temperature. 15. Pipette the organoid suspension repeatedly against the tube wall for 1 min (the suspension becomes viscous because of DNA released by dead cells). 16. Add 1 ml prewarmed Dispase and 50 μl DNase. Filter sterilize Dispase immediately before use because Dispase granules precipitate with cells and are toxic if present in the culture medium. 17. Pipette the cell suspension repeatedly against the tube wall for 2 min (aggregates should dissolve and the suspension should become clear; if not, add 50 μl of DNase and pipette for 1 more minute).
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18. Pass cells through a 40 μm cell strainer into a new 15 ml tube. 19. Spin at 1,400 rpm (~400 × g) for 3 min and remove the supernatant. 20. Resuspend cells in the selected culture medium, count cells, and plate as appropriate. 21. Incubate in a 37 °C, 5 % CO2, 5 % O2 incubator. 6.4 Infection of Mammary Epithelial Cells
The protocol below describes infection at an moi of 10, but it is often helpful to test a range of multiplicities, in case the titer was wrong or the cells are unusually sensitive to the virus. Always include a mock-infected well to check the efficiency of the antibiotic selection and an infected well that is not selected in antibiotic to check for toxicity of the virus. We normally grow primary cells in 5 % O2 but if using established cell lines this is not necessary. The viral suspension is in serum-free medium (OptiMEM). At commonly achieved titers (>107 IU/ml) this means
1
Introduction Lentiviral vectors are convenient tools to express transgenes in a wide variety of cultured cells. At first glance it might seem somewhat risky to use vectors derived from HIV as basic research tools, but the labs that conceived them were aware of the risks and went to great lengths to make them safe [1–4]. Additional information on safety is given in Notes 1 and 2. Minimally, you should familiarize yourself with level 2 biosafety precautions and make the requisite declarations to the authorities before starting to work with lentiviral vectors. Unlike transgenes in transfected DNA, transgenes in lentiviral vectors are immediately integrated stably into the genome of the target cell, greatly increasing the chance that they will be expressed stably. Unlike simple oncoretroviral or gamma retroviral vectors, lentiviral vectors can infect nondividing cells. The critical difference is that the cDNA produced by reverse transcription of lentiviral RNA is professionally imported into the nucleus, whereas cDNA from simple retroviruses must wait for the nuclear envelope to break down at mitosis to gain access to the chromosomes. This simplifies the protocol because it is not necessary to passage the target cells immediately after infection to make them divide.
Maria del Mar Vivanco (ed.), Mammary Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 1293, DOI 10.1007/978-1-4939-2519-3_8, © Springer Science+Business Media New York 2015
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A second advantage is that titers tend to be higher and virus can be frozen with minimal loss of titer. The vectors are normally pseudotyped with vesicular stomatitis glycoprotein (VSV G), which broadens the tropism because it works by inducing fusion with the plasma membrane. This works well for mammary epithelial cells, which are normally easy to infect with VSV G pseudotyped lentiviral vectors.
2
Overall Strategy The basic approach to make lentiviral vectors is to transfect 293T cells with a vector plasmid and two or three helper plasmids. The protocol below uses calcium phosphate transfection because it is cheap and highly effective in 293T cells. The transfection efficiency should be close to 100 %; if you find it difficult to achieve this, try commercial transfection reagents instead, such as Fugene (Promega).
3
Packaging Vectors Most labs use so-called second-generation plasmids, in which one helper plasmid contains all the lentiviral helper genes and a second helper plasmid contains VSV G. Other than tat and rev, the lentiviral accessory genes (vif, vpr, vpu, and nef) are all deleted from the helper plasmids. In third-generation systems the lentiviral genes are split between two plasmids, with the gag, polymerase, protease, and integrase genes on one plasmid and rev on another. In this configuration the tat gene is deleted, which means the vector plasmid requires a tat-independent promoter. A plasmid expressing VSV G is still required, meaning a total of four plasmids must be transfected. The third-generation system is considered safer but the need to transfect an additional plasmid may reduce the titer. In practice, most users choose the second-generation psPAX2 and pMD2.G packaging vectors developed by the Naldini and Trono labs (Fig. 1a, b, Addgene 12259 and 12260).
4
Lentiviral Expression Vectors There is a much wider choice of vector plasmids. In addition to deletion of all complete HIV genes, current vectors have partial replacement of the 5′-LTR by a heterologous promoter to make transcription in the packaging cell independent of tat; a deletion in the 3′-LTR to make both LTRs inactive as promoters after integration; and the presence of an RNase H-resistant central polypurine tract that primes reverse transcription and promotes nuclear import of
CMV enhancer Chicken beta actin promoter
ampR (bla)
actin intron
ColE1 origin SV40 ori psPAX2
HIV Gag
10703 bp
beta-globin polyA
HIV Protease HIV Tat/Rev HIV RRE HIV Polymerase HIV cPPT HIV Integrase
CMV promoter
ampR (bla)
beta globin intron pMD2.G pBR322 ORI
5824 bp
VSV-G beta globin polyA
Fig. 1 Lentiviral plasmids. (a) psPAX2 contains the main lentiviral genes required for packaging: gag, protease, polymerase, integrase, tat, and rev. (b) pMD2.G expresses VSV glycoprotein, which replaces the normal envelope protein. VSV G fuses directly with the plasma membrane, allowing the viral particles to infect cells indiscriminately. (c) pSD-82 is a typical lentiviral expression vector. The genomic transcripts made in the packaging cells initiate at a hybrid RSV/HIV promoter. The RSV enhancer is deleted in integrated proviruses because the 3′-LTR is copied to the 5′-LTR during reverse transcription. There is a deletion removing the enhancer in the U3 region in the 3′-LTR. This deletion is copied to the 5′-LTR after reverse transcription to create integrated proviruses with defective promoters in both LTRs (a so-called self-inactivating or SIN vector). This means the first transcripts from the integrated proviruses initiate at the human PGK promoter. Since this is located after the packaging signal (psi), no transcripts from the integrated provirus can be packaged. The transgene, ESR1, is located between two Gateway attB sites and expressed from the human PGK promoter. The WPRE located after the attB2 site increases the level of the ESR1 RNA. The puromycin resistance gene is expressed from the murine PGK promoter. The viral RNAs all terminate in the 3′-LTR. It is important to delete polyA signals from the transgene to avoid cleaving the 3′-end off the genomic RNA that is packaged in the viral particles
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pUC ORI
RSV LTR U3 HIV LTR RU5 psi RRE
ampR cPPT pSD-82 F1 ORI
hPGK promoter
9676 bp attB1
SV40 ORI HIV SIN LTR U3'RU5 puroR (pac) mPGK promoter
ESR1 orf attB2 WPRE
Fig. 1 (continued)
the preintegration complex. Many companies now sell lentiviral vectors but a cheap and convenient option for academic labs is to use vectors from Addgene. In addition to specific vectors that have been described in publications, Addgene contains an impressive collection of vectors deposited by Eric Campeau, many of which are based on the Gateway cloning system (http://www.addgene. org/Eric_Campeau/). The Campeau vectors systematically offer antibiotic resistance cassettes for puromycin, neomycin, hygromycin, blastomycin, and zeomycin, making it possible to select for multiple different vectors in the same cell. We have had difficulty selecting for hygromycin in primary mammary epithelial cells but the other markers all work well. The choice of promoter is dictated by the propensity of some promoters to become silenced more easily than others (cellular promoters are often better than viral promoters), and by the characteristics of the cells transduced (PGK works well in mammary progenitors and stem cells). The vector we have used most extensively in mammary epithelial cells has a human PGK promoter driving the transgene and a mouse PGK promoter driving the puromycin resistance gene (pac) (Fig. 1c) [5]. Note that the vector contains a Woodchuck hepatitis virus RNA stability element (WPRE) between the two expression cassettes, so the WPRE is only present in the RNA produced by the promoter driving expression of the transgene. This is a good idea in the sense that it increases the chance of the transgene being expressed well, but careful titration of puromycin is required to avoid killing the cells if they are infected at single copy. Many of the Campeau vectors have the same promoters in the same configuration and express transgenes well in mammary epithelial cells.
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Materials
5.1 Components for Lentiviral Production
293T/17 cells (ATCC CRL-11268). Dulbecco’s modified Eagle medium, 10 % FBS. Phosphate-buffered saline. 0.05 % trypsin/EDTA. TE buffer, pH 8.0. DNase/RNase-free water. Polylysine. 2.5 M CaCl2, 18.4 g for 50 ml; filter sterilize (0.22 μm) and store aliquots at −20 °C. 2 M HEPES, 52.6 g for 100 ml; filter sterilize (0.22 μm) and store aliquots at −20 °C. OptiMEM (Life Technologies); add 2 M HEPES to make OptiMEM 20 mM HEPES. HEPES-buffered saline (2× HeBS, Sigma 51558—50 ml). 10 cm tissue culture dishes. 15 and 50 ml conical base tubes. 0.45 and 0.22 μm syringe filters. Vivaspin 20 MWCO 100,000 (GE Healthcare #28-9323-63). 5 % CO2, 37 °C incubator. 1 % Virkon (50 g sachets, Dupont). 70 % (v/v) ethanol. Plasmids to make a simple GFP expression vector pRRLSIN.cPPT.PGK-GFP.WPRE (classic GFP expression vector, Addgene 12252). pMD2.G (for pseudotyping with VSV G protein, Addgene 12259). psPAX2 (contains the main HIV-1 helper genes: Gag, Pol, Tat, and Rev, Addgene 12260). Many other useful plasmids can be obtained from Addgene. See Notes 3 and 4 for information on construction and production of plasmids. All plasmid maxipreps should be “endotoxin-free” (Qiagen EndoFree or Nucleobond Xtra EF). For convenience, adjust the concentration of plasmids to 1 mg/ml.
5.2 Components for Titration of Viruses
Target cells (e.g., MCF7 cells). Cell culture medium (RPMI 10 % FCS for MCF7 cells). Viral suspension. Phosphate-buffered saline.
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0.05 % trypsin/EDTA. 6-Well plates. FACS tubes (polypropylene). 5 % CO2, 37 °C incubator. 5.3 Components for Isolation of Mammary Epithelial Cells
Cold DMEM/F12, phenol red-free, 2× Pen/Strep = minimal medium (MM). Phosphate-buffered saline. Collagenase A: Make 100 mg/ml in PBS, and store aliquots at −20 °C. Trypsin solution 0.25 %. Dispase: Make 5 mg/ml in PBS, filter sterilize (0.22 μm), and store aliquots at −20 °C. Deoxyribonuclease 1 (DNase): Make 1 mg/ml in PBS, filter sterilize (0.22 μm), and store aliquots at −20 °C. Cell strainer 40 μm. Sterile forceps. Sterile scalpels. Sterile chopping board. 10 cm cell culture dishes. 15 and 50 ml conical base tubes.
5.4 Components for Lentiviral Transduction of Mammary Cells
Single cells from reduction mammoplasty. Cell culture medium (according to individual lab preference). Viral stock. Phosphate-buffered saline. 0.05 % trypsin/EDTA. 6-Well plates. 5 % CO2, 37 °C incubator. Use 5 % O2 for primary cultures.
5.5 Components for RCL Test
Viral suspension. HIV genomic RNA (if available). 293T/17 cells (ATCC CRL-11268). Dulbecco’s modified Eagle medium, 10 % FBS. Phosphate-buffered saline. 0.05 % trypsin/EDTA. Trizol LS. RNeasy kit. 6-Well plates. 5 % CO2, 37 °C incubator.
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Reagents for routine molecular biology Superase inhibitor, 20 U/μl. DNase I (RNase-free) 10 U/μl. Restriction buffer M (Roche 11417983001). DNase/RNase-free water. 10 % SDS. Phenol/chloroform/isoamyl alcohol. NaOAc 3 M pH 5.5. Glycogen 20 mg/ml. Ethanol 70 and 100 % (molecular biology grade). Oligo-dT 0.5 mg/ml. dNTPs (10 mM each). Superscript II (reverse transcriptase, first-strand buffer, DTT). Sybr Green qPCR master mix 2× (Life Technologies 4344463). Forward primer PAXpolF: tggcagaaaacagggagatt. Reverse primer PAXpolR: tgcccctgcttctgtatttc. qPCR machine.
6
Methods Follow national biosafety level 2 guidelines when working with lentiviral vectors. If you are unsure what to do you are almost certainly not legal, so seek advice on local regulations from more experienced colleagues before starting the project. See Note 1 for additional information on biosafety of lentiviral vectors.
6.1 Production of Lentiviral Particles
293T cells grow rapidly and detach easily. Coating the dishes with polylysine helps to prevent the cells falling off the dish during medium changes. The protocol below differs from some classic protocols in that cells are plated 3 days before transfection. This is designed for a plate on Friday–transfect on Monday–harvest on Wednesday strategy. Despite the relatively high density at the time of transfection, the yields are excellent. 293T cells should be maintained in DMEM and 10 % FCS and split 1:10 twice a week. Take care always to passage the stock plates before the medium is exhausted. Replace the cultures at least every 3 months from frozen stocks that have been checked for mycoplasma. If none of the vectors contains GFP it is helpful to set up an additional transfection with GFP to check the efficiency of transfection. 1. Coat tissue culture plates with polylysine by briefly pipetting 5 ml of polylysine onto 10 cm plates.
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2. Plate 2 × 106 293T cells per 10 cm dish 3 days before transfection. 3. On the day of transfection, replace the medium with 5 ml DMEM and 10 % FCS. Cells should be about 80 % confluent when transfected (see Note 7). 4. For each 10 cm plate, prepare the following plasmid cocktails in 1.5 ml Eppendorf tubes: 4 μg pMD2.G (VSV-G viral envelope) 10 μg psPAX2 (packaging construct) 10–20 μg of your expression vector, adjusted according to the size of the plasmid (10 μg 10,000 bp) Add sterile water to 450 μl. 5. Add 50 μl CaCl2 to the plasmid cocktails and mix thoroughly. Prepare 15 ml Falcon tubes containing 500 μl 2× HeBS. 6. Add the plasmid cocktails drop by drop to the Falcon tubes containing the 2× HeBS while shaking them on a vortexer at low speed. 7. Wait for 5 min to allow the precipitate to form. 8. Add the precipitate to the cells by pipetting it drop by drop, trying to homogeneously cover the whole plate. Ensure even spreading of the precipitate by gently swirling the dish. Return the dish to the 37 °C 5 % CO2 incubator. 9. 6–14 h after transfection, remove the medium and replace it with 8 ml OptiMEM 20 mM HEPES. 10. 38–46 h after starting the transfection, harvest the medium, which now contains lentiviral particles. Centrifuge at 2,500 rpm for 3 min to remove debris, and then filter with a 0.22 μm syringe filter. The titer will drop if the medium becomes very acid, so it is not worth waiting an extra day before harvesting the virus in the hope that the yield will go up. 11. Optional: To concentrate the virus 10- to 100-fold, use a Vivaspin column (see Note 7). This is only possible if you used serum-free medium (OptiMEM) as described above (if you use FCS the filter will concentrate unwanted proteins from the serum and become clogged). Before you embark on extensive centrifugation of virus, check that you have taken reasonable steps to contain aerosols that might be released by leakage of viral supernatant. 12. Aliquot virus in freezing vials and store at −80 °C. 6.2 Titration of Lentiviral Vectors
Cells differ in their infectability, so viral titers are only strictly valid on the cell line used to calculate the titer. Since it is often not possible to perform a titration on the cells you are most interested
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in, you need to bear in mind that there may be a systematic error when you do the experiment (the effective multiplicity of infection [moi] may be higher or lower than you intended). See Note 5 for more information on calculating viral titers. 6.2.1 Titration by Antibiotic Selection
This is the simplest approach. It only gives a rough idea of the titer but this is good enough for most experiments. A dilution series is performed and the titer is based on finding the first well with no dead cells after antibiotic treatment. This well is conventionally taken to have been infected at an moi of 1. This approach works best for antibiotics that kill fairly quickly, in particular puromycin, blastomycin, and hygromycin. 1. Plate cells at a relatively low density to ensure that they can be left for a week without becoming overgrown. For MCF7 use 1 × 105 cells per well in 6-well plates, one plate per virus to be titered. 2. Infect cells. The following day, prepare six Eppendorf tubes each with a final volume of 1 ml medium containing: A. 1 μl virus B. 10 μl virus C. 100 μl virus D. 100 μl virus (control for toxicity of virus) E. No virus (control for toxicity of virus) F. No virus (control for antibiotic efficacy) 3. Replace the medium on the cells with the 1 ml dilutions in the Eppendorfs. 4. Incubate for 6 h, then remove the medium, and add 2 ml of fresh medium. 5. Antibiotic selection: 48 h after infection, remove medium and add 2 ml of fresh medium containing antibiotic to wells A, B, C, and F. Add 2 ml of fresh medium without antibiotic to wells D and E. The concentration of antibiotic required depends on the cells, so before testing the virus you should titrate the antibiotic to find the concentration that kills uninfected cells in a reasonable time—for MCF7 it is likely to be around 2 μg/ml puromycin, 200 μg/ml hygromycin, 5 μg/ml blasticidin, 750 μg/ml G418, and 1 mg/ml zeomycin. The time to kill 100 % of the cells should be around 3 days for puromycin, 5 days for blasticidin and hygromycin, 10 days for zeomycin, and 14 days for G418. 6. Calculate viral titer. Observe the cells and score them when there is obvious death in well F. This will be 2–3 days for puromycin, and up to 1 week for the other antibiotics. G418 is so slow that it may be hard to interpret the results because the
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cells are too confluent. The goal is to find the well with the lowest amount of virus giving no death. Compare wells D and E to check that death is due to antibiotic rather than a direct toxic effect of the virus. Titer (infectious particles/ml) = number of cells at infection/ volume of virus in ml in the first well with no death. If you used the amounts above, the titers will be: 1 × 105/0.001 = 1 × 108 infectious particles/ml if there is no death in well A 1 × 105/0.01 = 1 × 107 infectious particles/ml if there is no death in well B 1 × 105/0.1 = 1 × 106 infectious particles/ml if there is no death in well C Good titers are 107–108 infectious particles/ml in unconcentrated viral supernatant. This approach will slightly underestimate the titer (see Note 5 for the reason why). 6.2.2 Titration by Flow Cytometry
It is possible to titer viruses accurately by flow cytometry provided they express a fluorescent protein such as GFP. It is the percentage of cells positive not the intensity of expression that matters. 1. Plate 1 × 105 MCF7 cells per well in 6-well plates, one plate per virus to be tested. Seed two additional wells that will not be infected, so you can use one to count the exact number of cells at the time of infection and the other as a negative control to set the flow cytometer gates. 2. Infect cells. The day after plating the cells, prepare Eppendorf tubes containing serial twofold dilutions of virus. In the first tube mix 64 μl virus with 1,934 μl medium. Then set up five tubes containing 1 ml medium and make serial dilutions by transferring 1 ml sequentially to each tube. 3. Remove the medium from the cells and add 1 ml of each virus dilution per well. Harvest one uninfected well to count the number of cells at the moment of infection (this lets you correct for the plating efficiency and for cells that have divided since they were plated). 4. Incubate for 6 h, then remove the medium, and add 2 ml of fresh medium. 5. Three days after infection, aspirate the medium, add 0.5 ml trypsin/EDTA to each well, including the control uninfected well, and incubate at 37 °C until the cells detach. 6. Add 1 ml medium to neutralize the trypsin, pipette up and down to ensure that the cells are dissociated, and then transfer the suspension to 5 ml polypropylene FACS tubes.
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7. Pellet the cells by centrifuging for 5 min at 1,500 rpm and discard the supernatant. Optional: If your flow cytometry facility does not accept cells recently transduced with level 2 viruses, kill residual virus by resuspending the pellet in 0.5 % formaldehyde in PBS and incubating at room temperature for 5 min. Then pellet the cells again by centrifuging for 5 min at 1,500 rpm and discard the supernatant. Note that formaldehyde is compatible with GFP fluorescence but may abolish the fluorescence of other markers. 8. Resuspend the cells in 200 μl of PBS, store them at 4 °C, and perform flow cytometry within a few hours. Use the uninfected control well to set the gate for background fluorescence. 9. Record the percentage of infected cells in each well. 10. In Excel, make a table with the volume of virus in ml in one column and the percentage of fluorescent cells in another column. Add a column called “moi” in which you enter a formula for the “negative log of the fraction of cells not infected.” The formula should look like this: = -LN ( (100 - POS ) / 100 ) where POS refers back to the column with the percentage of fluorescent cells, and LN() takes the natural log. The “moi” column now contains the moi achieved with the specified amount of virus. 11. Make a graph with volume of virus in ml on the x-axis and “moi” on the y-axis. Exclude any obvious outliers (they may occur at the most extreme virus concentrations), then invite Excel to draw a straight line through the points, and ask it to display the slope and intercept of the line. The viral titer in infectious units per ml is given by titer = ( slope ´ cell number ) / (1 - intercept ) where cell number is the number of cells present in the well at the time of infection. See Note 5 for additional information on calculation of titers. 6.2.3 Titration by p24 ELISA
It is possible to titer lentiviral vectors by ELISA for p24, a capsid protein derived from the gag polyprotein. This approach measures the amount of p24 in pg, which depends on the number of physical virus particles. The ratio of infectious units to physical particles (typically 1 in 100 to 1 in 1,000) is used to assess the quality of viruses, for example to detect batch-to-batch variation in the quality of preps destined for clinical use. Since the quality of typical
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academic lab virus preps is highly questionable, it is necessary to make some very large assumptions when extrapolating from physical titers to infectious titers. The approach is only useful if you have a burning desire to know the titer and there is no other way to do it (no antibiotic marker, no fluorescent marker, no transgene that can be detected in transduced cells by IF or IHC). When forced to measure physical titers we use the Innotest HIV Antigen mAb P24 ELISA kit (#80563, http://www.fujirebio-europe.com) according to the manufacturer’s instructions. 6.3 Isolation of Mammary Epithelial Cells
The protocol can conveniently be split into two parts separated by the freezing of the organoids. 1. Place reduction mammoplasty tissue on chopping board. 2. Remove yellow fatty tissue by scraping it off with a scalpel. 3. Shear and chop the remaining tissue using two scalpels until it reaches a mushy consistency. 4. Add 1 g tissue to 10 ml MM + 100 μl Collagenase in 50 ml tube. 5. Incubate at 37 °C with agitation (roller) for 3–16 h. 6. Spin at 1,400 rpm (~400 × g) for 4 min. 7. Wash pellet twice with warm PBS and 2 % FCS. 8. Resuspend pellet (“organoids”) in 2 ml freezing medium (10 % DMSO, 90 % FBS; or Cryo3). 9. Aliquot into cryotubes and freeze in isopropanol box (Mr. Frosty, Nalgene) at −80 °C overnight and then transfer to liquid nitrogen. 10. Thaw organoids and transfer to 15 ml tube containing 4 ml prewarmed MM medium. 11. Spin at 1,400 rpm (~400 × g) for 1 min. 12. Eliminate the supernatant carefully. 13. Add 500 μl prewarmed trypsin 0.25 % and resuspend organoids. 14. Incubate for 2 min at room temperature. 15. Pipette the organoid suspension repeatedly against the tube wall for 1 min (the suspension becomes viscous because of DNA released by dead cells). 16. Add 1 ml prewarmed Dispase and 50 μl DNase. Filter sterilize Dispase immediately before use because Dispase granules precipitate with cells and are toxic if present in the culture medium. 17. Pipette the cell suspension repeatedly against the tube wall for 2 min (aggregates should dissolve and the suspension should become clear; if not, add 50 μl of DNase and pipette for 1 more minute).
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18. Pass cells through a 40 μm cell strainer into a new 15 ml tube. 19. Spin at 1,400 rpm (~400 × g) for 3 min and remove the supernatant. 20. Resuspend cells in the selected culture medium, count cells, and plate as appropriate. 21. Incubate in a 37 °C, 5 % CO2, 5 % O2 incubator. 6.4 Infection of Mammary Epithelial Cells
The protocol below describes infection at an moi of 10, but it is often helpful to test a range of multiplicities, in case the titer was wrong or the cells are unusually sensitive to the virus. Always include a mock-infected well to check the efficiency of the antibiotic selection and an infected well that is not selected in antibiotic to check for toxicity of the virus. We normally grow primary cells in 5 % O2 but if using established cell lines this is not necessary. The viral suspension is in serum-free medium (OptiMEM). At commonly achieved titers (>107 IU/ml) this means
