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Phoenix - Helper free protocol

The following protocol is an adaptation of a review in press, it has been changed to accomodate the Phoenix systems protocol developed by Achacoso and Nolan. This is one of four transfection protocols you can find in this web page set.

IMPORTANT NOTE

The viral supernatants produced by these methods might,depending upon your retroviral insert, contain potentially hazardous recombinant virus. The user of these systems must exercise due caution in the production, use and storage of recombinant retroviral virions, especially those with amphotropic and polytropic host ranges. This consideration should be applied to all genes expressed as amphotropic and polytropic pseudotyped retroviral vectors. Appropriate NIH and other regional guidelines should be followed in the use of these recombinant retrovirus production systems.

The user is strongly advised NOT to create retroviruses capable of expressing known oncogenes in amphotropic or polytropic host range viruses.

Rapid Production of High Titer, Helper-free Retroviruses using Transient Transfection

Warren S. Pear*, Martin L. Scott*, and Garry P. Nolan#


#Dept. of Molecular Pharmacology, Dept. of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305
*Dept. of Biology, Massachussetts Institute of Technology, Cambridge, MA 02139

1. Introduction

Retroviral gene transfer is presently one of the most powerful techniques for introducing stably heritable genetic material into mammalian cells (reviewed in (1)). One serious drawback of this technique, however, has been the difficulty in readily producing high titer recombinant retroviruses. For many applications, such as infecting rare target cells or the majority of cells in tissue culture, the recombinant virus titer must be at least 10^6 infectious units/ml. Although one can usually obtain high titer mixtures of recombinant and replication-competent retroviruses in a relatively short time, many applications such as cell marking studies or studying genes in vivo demand freedom from replication competent virus.

A milestone in gene transfer technology was the creation of cell lines that could package retroviral RNA's into infectious particles without the concomitant production of replication competent virus (2), (3), (reviewed in (4), (5)). In what became the prototype for creating helper-free retrovirus packaging cells, the retroviral structural gene products (gag, pol, and env) were provided in trans by separating them from the elements required for packaging retroviral RNA into virions. The first ecotropic retroviral packaging line, Psi-2, was created by stably introducing into NIH3T3 cells an engineered retroviral DNA genome from which the RNA packaging signal, termed y, had been removed (2). Production of high titer, helper-free, infectious virus required the subsequent stable introduction of a retroviral vector containing theðy retroviral packaging site, transcription and processing elements, and the gene of interest into the Psi-2 cells . Retroviral particles produced in this way could infect target cells and transmit the gene of interest, but not replicate, because genomic information encoding the packaging proteins was not carried in the packaged retroviral particles. This original strategy has subsequently been modified by a number of investigators to decrease the risk of producing replication competent virus and to increase viral titers (see reviews by (4), (5)). Although infectious retrovirus is produced within 48 hours following transfection of these packaging lines, the infectious titer generated by these cells is generally low (10^3-10^4 /ml), necessitating identification of those clones that produce retroviruses at higher titer. This is accomplished by selecting single cell clones from the transfected population, then testing each for its ability to produce virus (Fig. 1a). Under optimal circumstances, this requires 1-2 months and must be repeated for each different construct. During theðprolonged selection process, gene(s) encoded by the retroviral vector may inhibit growth of the packaging line, favoring the outgrowth of clones that express low levels of virus; thereby, making it difficult to identify clones continuously producing retrovirus at a high titer. Although the biochemical basis for this effect is often unclear, the inability to produce high titer infectious retroviruses expressing certain genes, such as members of the abl and rel families, has been experienced by a number of investigators.

To minimize potential toxic effects of the introduced gene products on the packaging cell lines, several groups have devised strategies based on transient retroviral production (6), (7), (8). This approach has the added benefit of markedly reducing the time and effort required to produce high titer retroviral stocks. Although the first step in both stable and transient retroviral production is transfection of the retroviral construct into the packaging cell line, at 48 hours after transfection, the infectious retroviral titers are several logs higher using the transient production methods.

Several factors may account for the ability of the transient retroviral production methods to yield viral supernatants with infectious titers in the range of 10^5-10^7/ml. These include both the properties of the parent cells themselves and changes engineered into them to augment transient production. Unlike previous retroviral packaging cell lines based on either murine or avian cells, the transient approaches utilize cells derived from either primates or humans. These cells offer several potential advantages. First, they are much more transfectable using simple CaPO4 based protocols. Second, they support very high level expression of genes introduced under control of a number of promoters, such as those derived from CMV. Third, they contain fewer endogenous retroviral loci whose products could interfere with infectious virus assembly and release (9). In addition, they have been previously modified to contain viral gene products, such as adenoviral E1A or polyoma large T antigen, with properties that can be exploited to further enhance expression of new constructs.

The strategy described by Landau and Littman (6) uses transient co-transfection of the SV40-transformed African green monkey renal cell line, COS-7, with two modified retroviral plasmids, both containing the SV40 origin of replication. In addition to this viral element, one construct contains the MLV LTR, retroviral packaging site, SV40 origin of replication (ori), and DNA element(s) of interest, whereas the second construct encodes retroviral core (gag), polymerase(pol), and envelope (env) proteins but lacks a retroviral packaging site. Virus stocks with titers as high as 10^5 infectious units/ml have been obtained with certain recombinant constructs by using this approach. The ability to obtain titers in this range, depends, in part, on large T antigen-dependent amplification of the plasmids containing the SV40 ori, which can enhance infectious recombinant titers by up to 2 logs (6). Although these authors do not report the detection of replication competent virus, the vectors used in this study could produce such genomes after only one recombination event.

Both of the other transient transfection strategies (7), (8) use the 293 cell line, an adenoviral transformed human embryonic kidney cell line (10) as the basis of their packaging systems. In the approach described by Pear et. al. (7), 2 different plasmids encoding the retroviral structural genes, in trans, were stably incorporated into the 293T cell line, a variant of 293 cells into which a temperature sensitive SV40 large T antigen had been introduced (11). As the retroviral structural genes are encoded on two different plasmids and contain additional mutations, at least 3 recombination events are necessary to generate replication competent retroviruses (12). Stable incorporation of these plasmids is important for preventing helper virus formation since transient introduction of these plasmids together with a Moloney-based retroviral vector yielded replication competent virus (7). Using this system, all constructs tried by the authors have produced retroviral titers greater than 10^6/ml. This includes not only relatively non-toxic genes such as those encoding b-galactosidase (bgal) and G418 resistance, but also members of the abl and rel families of proto-oncogenes from which we were unable to identify stable high titer producing clones utilizing the stable production approach (7). These viruses have been used to infect rodent tissue culture cells and murine hematopoietic progenitors with no detectable generation of helper virus (7). Although retroviral vectors containing an extended packaging site (13) give approximately a 2-fold increase in titer, infectious titers greater than 10^6/ml have been obtained with all of the retroviral vectors tried by the authors including pGD(14), pBABE (15), MFG (16), and pBND (D. Turner and C. Cepko, unpublished, described in (7)). We have observed no enhancement of titer using retroviral vectors containing an SV40 ori, despite the fact that a 293 cell variant (293T; (11) ) was chosen as the starting material for the packaging cell lines.

The system described by Finer et. al. (8), termed kat, also utilizes 293 cells. However, unlike the cell lines of Pear et. al. (7), the kat system requires co-introduction of both a plasmid encoding the retroviral structural genes and a plasmid encoding the retroviral vector. The constructs utilized in the kat system have been further modified to minimize the creation of replication competent retroviruses. In general, the titers obtained using kat-produced retroviral supernatants are similar to those found by Pear et. al (7). It is unclear whether further advantage would result from the creation of a packaging cell line by stable introduction of the kat constructs. Another question is whether all MuLV-based retroviral vectors will function in the kat system or whether only the re-engineered kat vectors function well in this system.

In summary, both the methods described by Pear et. al. (7) and Finer et. al. (8) give rapid production of high titer, helper free viruses. These methods are particularly useful in creating retroviral stocks containing genes that are toxic to stable producer lines. Also the ability to rapidly produce high titer retroviral stocks offers novel possibilities for using these viruses, for example in the creation of retroviral cDNA libraries.

The remainder of this chapter will focus on the use of the packaging cell lines developed by the Achacoso and Nolan for rapid production of helper-free retroviruses with ecotropic, amphotropic, and polytropic host ranges. The basic 293 cell transfection protocols are likely to work equally well with the kat system plasmids, or other complementation systems which provide the requisite packaging proteins and genomic material to package. Procedures are described for optimizing transfection conditions and performing infections of adherent and non-adherent cell types. The advantages of these transient retroviral production methods should facilitate and extend the use of retroviral gene transfer technology. In addition to producing helper-free ecotropic and amphotropic retroviruses, methods are presented for rapidly pseudotyping retroviral virions with alternative envelope proteins.

1.1 Rapid Pseudotyping Of Moloney Retroviral Virions With Vesicular Stomatitus Virus G-Glycoprotein

Caldwell and Nolan have successfully produced high-titer VSV-G pseudotyped virions for infection of human and non human cell types. The production of VSV-G to pseudotype retroviral virions, demonstrated by Burns et. al. (21), is shown here in an adaptation of the transient retrovirus system using the Anjou cell line. A more detailed description of the use of this pseudotyping approach to infect several different human cell lines, mouse cells, fish cells, and insect cells will be published elsewhere (J. Caldwell, J. Lorens, P. Achacoso, and G. Nolan, paper in preparation and unpublished results). The method outlined in this chapter for pseudotyping does not require the selection of a stable producer clone, as required by the procedure of Burns , and results in the production of recombinant virus three days after transfection of the construct. The VSV-G pseudotyped viruses produced in this fully transient system can be concentrated and used to infect multiple human and non-human cell types. It is expected that this approach will also be useful for pseudotyping retroviral virions with designer envelope proteins that confer cell-type specific infection of target cells. Such specificity should be of therapeutic importance in numerous clinical settings. The main advantage of VSV-G pseudotyping is the stability imparted on the retroviral virion by the VSV-G envelope. This stability allows for concentration of virion stocks by centrifugation (multiple rounds can be employed for continued concentration) to very high titers. A second advantage is that the VSV-G target epitope is phosphatidylserine (22), a widely expressed lipid in higher eukaryotes. It is the broad expression of this latter target epitope that expands the potential host range of retrovirus-mediated gene delivery.

2. Materials

All solutions are prepared using double distilled water. For reagents used in tissue culture, it is recommended that they are prepared in disposable plastic labware. When possible, it is best to order reagents which have been tissue culture tested by the manufacturer.

2.1. Growth Medium(GM) for 293T Cells and Derivatives. The following are added directly to DME to give the indicated final concentrations: 10% heat inactivated fetal bovine serum , 100 U/ml Penicillin, 100 U/ml Streptomycin, 2 mM L-Glutamine.

2.2 Freezing Medium: 90% heat-inactivated fetal calf serum, 10% DMSO

2.3. Chloroquine: 25 mM chloroquine stock solution prepared in either PBS or GM and filtered through a 0.2 µM filter and stored at -20°C.

2.4 Transfection Reagents (a). 2 x HBS: 50mM HEPES, pH 7.05; 10 mM KCl; 12 mM Dextrose; 280 mM NaCl; 1.5 mM Na2HPO4 (FW 141.96). The final pH of the solution should be 7.05 +/- 0.05. Filter through a 0.2 µM filter, aliquot, and store at -20°C. Try to avoid multiple freeze/thaw cycles. To thaw, warm to room temperature and invert or vortex the tube to achieve uniform mixing. Although it is unclear why this occurs, the ability of the 2xHBS solution to produce working CaPO4 precipitates deteriorates after 6 months to one year, even when the 2xHBS solution is stored at -20°C. (b). 2 M CaCl2: Prepare a 2M solution and filter through a 0.2 µM filter, aliquot, and store at -20°C.

2.5 Standard fibroblast medium (SFM) for growth of NIH3T3 cells The following are added directly to DME to give the indicated final concentrations: 10% heat inactivated donor bovine serum, 100 U/ml Penicillin, 100 U/ml Streptomycin, 2 mM L-Glutamine.

2.6. Polybrene: The stock concentration is 4 mg/ml (dissolved in PBS and subsequently filtered through a 0.2 µM filter and stored at either 4°C or -20°C).

2.7. Constructsused in VSV Pseudotyping pME-VSV-G. The cDNA for the VSV-G polypeptide of Vesicular Stomatitus virus (gift of J. Rose, Yale) was inserted into the vector pME18S (gift of T. Kitamura, DNAX) under the transcriptional control of the SRa promoter element (an SV40/HTLV-1 hybrid promoter). MFG-lacZ is a retroviral vector expressing lacZ under the control of the MLV LTR (gift of Richard Mulligan, Whitehead Institute).

2.8 Packaging Cell Lines

The 293T/17, Phoenix-gp, Phoenix-Eco, and Phoenix ampho cell lines are available for non-commercial use through the Nolan laboratory. In order to obtain the cell line(s), it is first necessary to complete and return a material transfer agreement. The material transfer agreements can be obtained from this web site.

For commerical use, you must contact Mona Wan, Office of Technology Licensing, Stanford University, 415-324-7203. Any questions regarding receipt of your commercial transfer agreement should be addressed to this office.

3.1. Growing and Freezing the Cells

293 cells and the packaging lines derived from these cells are carried in 293 Growth Media (GM-see Materials) and grown in a 37°C degree incubator containing 5% CO2 . To split and passage the cell lines:

1. Gently rinse x 1 with PBS (without Ca++ or Mg++).

2. Trypsinize (.05% trypsin/0.53 mM EDTA) until the cells easily detach and can be readily pipetted into a single cell suspension (see Note ).

3. Trypsinization is quenched with GM prior to subculturing in fresh medium.

It is suggested that the cells not be split at densities more dilute than 1:5 in order to maintain the uniformity of the cells in culture and minimize the outgrowth of clonal variants. The cells should not be allowed to become overconfluent, as this leads to the formation of cell clumps in culture which can cause uneven cell distribution after replating and result in less efficient transfection.

3.2 Freezing 293 cells and Derivatives:

To assure viability of the cell line, it is recommended that the cells are frozen prior to confluence.

293 cells and their derivatives are much less adherent than NIH3T3 cell lines and easily detach from the tissue culture dishes after approximately 30 seconds of treatment with trypsin at room temperature.

1. To freeze: Wash, trypsinize, and quench cells as described in 3.1.

2. Centrifuge the cells at 500 x g for 5 min.

3. Remove the media and add 1 ml of freezing solution (see Materials) per 10^6 cells.

4. Transfer to a 2 ml cryogenic vial. 5. Place the freezing vial at -70 °C overnight and transfer to liquid nitrogen on the following day.

3.3. Thawing 293 Cells And Derivatives.

It is important to freeze multiple (50-100) vials of each producer cell line after first receiving and expanding them. This will ensure a ready supply of backup vials to allow for uniform virus production over several years. If the cells are to be carried in selective media, this should not be applied until after the first passage.

1. Remove one vial from liquid nitrogen and thaw rapidly at 37°C.

2. Immediately add 1 ml GM to the freezing vial and gently transfer this solution to a 15 ml sterile conical screw cap tube.

3. Add 2 ml of GM and gently mix the tube to allow for osmotic equilibration.

4. Add 10 ml of GM, close the tube, invert several times and spin cells at 500 x g for

5 minutes. 5. Remove the supernatant, resuspend cell pellet in GM, and transfer to a 10 cm tissue culture dish.

3.4. Transfecting the Packaging Cell Lines:

Unless otherwise noted, all conditions are described for 60 mm plates.

1. Plate 2.5 x 10^6 cells per 60 mm plate in 4 mls of GM 24 hours prior to transfection. The dish should be approximately 80% confluent prior to transfection.

2. Just prior to transfection, change the medium to 4 mls of GM containing 25 uM chloroquine.

3. In an eppendorf tube, prepare the transfection cocktail by adding 6 to 10 µg DNA to H2O such that the final volume is 438 µl. Add 62 µl 2M CaCl2 to the DNA/H20 (see Materials). Add 500 µl 2X HBS (pH 7.05) by bubbling (see Materials and Note ). Immediately (within 1-2 minutes) add this solution to the cells and gently agitate to insure uniform mixing. Return the cells to the incubator.

4. Approximately 10 hours after adding the chloroquine containing medium, remove the medium and gently replace with fresh GM. (If chloroquine was not used in Step 3, skip to Step 5)

5. Approximately 16-24 hours prior to harvesting the retroviral supernatant, remove the medium, and gently add fresh GM.

6. Harvest the retroviral supernatant 48 hours post-transfection (see Note and Section 3.5).

It is recommended that during initial set up, the user optimize the system by using a retroviral vector expressing an easily assayable marker such as lacZ or a cell surface protein. During optimization, one should check for transfection frequency of the producer clone and test infection rate of target cells. Tests for transfection and infection frequencies using a bgal based system can be readily measured by bgal staining or FACS staining for bgal activity (see references in (7) for methods describing the latter procedures). Only when one is satisfied with the transfection conditions and infection rates should one proceed to using vectors with no readily assayable marker. It should be possible to scale up the protocols.

The initial plating of the cells may be the most important step in successfully obtaining high retroviral titers. It is extremely important that the cells are not overly clumped and are at the correct density. Unlike NIH3T3-derived cell lines, the 293-derived packaging cell lines do not readily form well-spread monolayers. Instead, they tend to clump before confluence, and if the clumping is excessive, the cells will never reach confluence during the 48-72 hour period following transfection. In order to prevent clumping, it is essential that the cells are extremely healthy prior to plating. If they are overconfluent, it may be necessary to split them 1:2 or 1:3 for several passages prior to plating for transfection. In addition, the cells are much less adherent than murine fibroblasts and should be handled very gently when washing and changing medium. For consistency, it is important to count the cells rather than estimating the split. The above cell number is optimized for MFG-lacZ. Expression of other inserts may be detrimental to the growth of the cells. This effect may be noted by failure of the packaging cell line to reach confluence by 48-72 hours post-transfection. If this occurs, it may be necessary to plate more cells prior to transfection. For example, with constructs expressing either fas or P210bcr/abl, it is necessary to plate 3.0 x 10^6 cells per 60 mm plate 24 hours prior to transfection. In general, the cells should be plated at a density so that they are 95-100% confluent at 24 hours post-transfection (for an additional discussion of this issue, see Notes 10 and 12).

The addition of chloroquine to the medium appears to increase retroviral titer by approximately two fold. This effect is presumably due to the lysosomal neutralizing activity of the chloroquine (23). It is extremely important that the length of chloroquine treatment does not exceed 12 hours. Longer periods of chloroquine treatment have a toxic effect on the cells causing a decrease in retroviral titers. The range for chloroquine treatment is 7-12 hours with 9-10 hours of treatment giving the best results. For purposes where achieving maximal retroviral titer is not necessary, such as when comparing the relative titers of different constructs, it may be preferable to omit chloroquine treatment. If chloroquine is not used, it is unnecessary to change the medium prior to transfection. On some occasions, we have obtained slightly improved transfection efficiencies by adding the chloroquine to a 1:1 mixture of 293 conditioned media (obtained from any of the 293-based cell lines) and fresh GM.

It is important that the pH of the Hepes be adjusted to 7.05 (within .05 units). Although, we generally add the HBS to the DNA/CaCl2 solution by bubbling, equivalent results can be obtained by adding the HBS to the DNA/CaCl2 solution and immediately inverting the tube. The HBS/ DNA/CaCl2 solution should be added to the cells within 1-2 minutes of preparation. It is not only unnecessary to wait for the formation of a visible precipitate, but waiting this long (15-30 minutes) may have a detrimental effect on transfection efficiency and subsequent retroviral titers. In addition, the presence and/or amount of precipitate that one visualizes following transfection is not a reliable indicator of transfection success. We have used DNA prepared by both cesium chloride gradients and several commercial kits and have not found significant differences among titers between the different preparation methods. It is unnecessary to perform additional phenol or precipitation steps prior to using the DNA (which is stored in TE (8.0) at -20°C). Up to a point, transfection efficiency and retroviral titers increase with increasing amounts of input DNA. The benefit of increasing the amount of input DNA must be weighed against our findings that this appears to have a direct toxic effect to the cell lines. If it is found that the amount of DNA is toxic to the cell line, it may be necessary to decrease the amount of input DNA or increase the number of plated cells. In some experiments, we have introduced up to 15 µg of dna to a 60 mm plate during transfection. Incubator CO2 concentrations which are outside the range of 4.5%-5.5% may adversely affect transfection efficiency.

In order to increase the relative retroviral titer, decrease the volume in this step to 3.0 mls/60 mm dish. depending upon the gene expressed from the introduced construct, the producer cells may be readily dislodged from the culture dish. It is therefore important to add the media to the side of the dish, taking care not to disturb the adherent cells.

3.5. Harvesting the Retroviral Supernatant

1. When the retroviral supernatant is ready for harvesting , gently remove the supernatant and either filter through a 45 µM filter or centrifuge x 5 min at 500 x g at 4°C to remove living cells. If the retroviral supernatant is to be used within several hours, keep on ice until it is used. Otherwise, the retroviral supernatant may be frozen, resulting in a minimal loss of viral titer (see Note ).

2. To freeze the retroviral supernatant: either snap freeze the tube in liquid nitrogen or place the tube in dry ice. The frozen samples are stored at -70 °C. To thaw the frozen samples: warm for a minimal period of time at 37 °C. The retroviral supernatant is ready for immediate use in subsequent experiments.

The packaging cells should be nearly confluent by 24 hrs post-transfection. under these circumstances, the retroviral supernatants should be collected at 48 hours post-transfection. If the cells are not confluent by this point, it may be necessary to wait until 72 hrs. With the transient transfection methods, we find that retroviral titers drop if supernatant is harvested after 72 hours post-transfection. If the cells are not confluent by 72 hours, decrease the amount of input DNA and/or increase the number of initially plated cells with the goal of obtaining a confluent plate by 48 hours post-transfection. In some experiments, we have used conditions where the plate is almost confluent at the time of transfection and have obtained good results. It is necessary to wait at least 36 hours post-transfection to obtain high titer retroviral supernatants. At this point, it may be possible to harvest the supernatant every 12 hours up to 72 hours without a significant loss in titer. If a retroviral vector containing an easily assayable marker, such as bgal, is used, it is possible to stain or FACS the cells 48 hours post transfection to test the success of the transfection. When initially optimizing the system, transfection efficiencies should approach at least 50%.

Freezing does not appear to cause more than a 2-fold drop in titer, as long as the cells do not undergo more than one freeze/thaw cycle. If the cells undergo more than one freeze/thaw cycle, there is a significant drop in retroviral titer.

No difference in infection efficiency was found between 4 µg/ml Polybrene and 8 µg/ml Polybrene. If 8µg/ml Polybrene is used, however, it is suggested that the Polybrene is left on the cells for 3-5 hours since the higher Polybrene concentration is toxic to the NIH3T3 cells. At 4 µg/ml Polybrene, no toxicity to the fibroblasts is observed; however, a 5 ml infection cocktail is recommended to prevent dehydration.

3.6. Infection of Adherent Fibroblasts

1. approximately 12-18 hours prior to infection, plate 5 x 10^5 NIH3T3 cells in SFM (standard fibroblast medium) on a 100 mm plate.

2. Prepare a 3 mls infection cocktail consisting of: i) retroviral-containing supernatant (either fresh or thawed), ii) Polybrene at a final concentration of 4 µg/ml, and iii) SFM.

3. Remove the SFM from the NIH3T3 cells and add the 3 mls infection cocktail to the cells.

4. Return to the incubator for at least 3 hours (see Note ).

5. Add 7 mls of SFM to the cells .

6. Harvest (stain, neo select, etc.) the infected cells at 48 hours post infection.

3.7. Infection of Non-adherent Cells by Addition of Retroviral Supernatant (see Note )

The conditions described are for infecting in 60 mm plates.

1. Prepare an infection cocktail consisting of: a) the medium in which the target cells are grown, b) retroviral supernatant and c) Polybrene (2 µg/ml) such that the total volume is 3 mls (see Note ).

2. Centrifuge exponentially growing target cells at 500 x g for 5 minutes. Remove supernatant and resuspend the cells in the infection cocktail at a concentration of 10^5-10^6 cells per ml.

3. Twenty four hours post-infection, centrifuge the cells (500 x g for 5 minutes) and resuspend in the appropriate media for normal growth of the target cells (see Note ). Allow the cells to grow for an additional 24-48 hours before drug selection or other assays, such as staining for lacZ activity.

When working with non-adherent cells, one has the choice of infecting by adding the retroviral supernatant directly to the cells or co-cultivating the non-adherent cells with the retroviral producer cells. The advantage of the latter is that there is ongoing retroviral production; however, this must be weighed against the disadvantage of harvesting producer cells together with the target cells. Although we have not tried, it may be possible to minimize this problem by irradiating or mitomycin C treating the producer cells prior to co-cultivation. In general, we have obtained higher infection frequencies by co-cultivation.

For many non-adherent cells, achieving an optimal infection requires growth in the appropriate medium. Because 293 cells and their derivatives appear to tolerate many different medium bases and serum types, it is possible to alter the medium at 24 hours post transfection (see Section 3.4, Step 5) so that the resulting retroviral supernatant will be harvested in the appropriate growth medium. When infecting with supernatants derived from 293 cells or derivatives , it should be remembered that these cells may provide a different cytokine/growth factor milieu than the NIH3T3-derived producer cells. A careful analysis of factor production by these cells has not been performed.

3.8. Infection of Non-adherent Cells by Co-cultivation with Retroviral Producing Cells.

Conditions are described for 60 mm plates.

1. Transfect the 293 cells or derivative cell lines as described in 3.4 Steps 1-4.

2. Twenty four hours post-transfection, prepare a 3 ml infection cocktail consisting of i) Polybrene at a final concentration of 2 µg/ml, ii) 1 ml of fresh or freshly thawed retroviral supernatant, and iii) the non-adherent cells at a density of 10^5-10^6 cells per ml in the appropriate media for normal growth of the target cells (see Note 16). Remove the medium from the cells and gently add the infection cocktail to the cells (Add the cocktail to the side of the plate rather than directly to the cells). Return the cells to the incubator.

3. Forty eight hours post-infection, gently remove the medium which will contain many non-adherent cells, and transfer to a conical tube. Centrifuge for 5 min at 500 x g (see Note ). Remove the supernatant and gently resuspend the cell pellet in a freshly prepared infection cocktail as described in Step 2.

4. With extreme care to avoid disruption of the adherent cells, add the infection cocktail (in which the non-adherent cells have been resuspended) to the wall of the plate (rather than directly to the cells). Return the plate to the incubator.

5. Twenty four hours later (72 hours post-transfection), remove the non-adherent cells from the dish by gently pipetting (see Note ).

6. Centrifuge the cells (5 min x 500 x g) and resuspend in the appropriate media for normal growth of the target cells . Allow the cells to grow for an additional 24-48 hours before drug selection or other assays, such as staining for lacZ activity.

At this point, it is important that disturbance of the packaging cells is minimized. Use extreme care when removing the non-adherent cells from the packaging cells and do not wash the plate at this step. Also, it is unimportant to remove all of the non-adherent cells at this step. The purpose of this step is to add fresh medium and retroviral supernatant without losing non-adherent cells. Sufficient residual media remains on the plate to maintain the cells during the short centrifugation step described in 3.8, Step 3. With longer centrifugation times, return the culture plates to the incubator.

The plate may be washed at this step, however, extreme care should be used so that the adherent cells do not detach. At this step, one is trying to achieve maximal removal of non-adherent cells. With this procedure, contamination by packaging cells is often less than 10%.

3.9 Rapid Pseudotyping Of Moloney Retroviral Virions With Vesicular Stomatitus Virus G-Glycoprotein.

1. Eight hours prior to transfection, plate the Anjou cells at 3 x106 cells per 60 mm plate in a 1:1 mixture of Anjou cell conditioned media and fresh GM (see Note ).

2. At eight hours post-plating, add chloroquine to 25 uM and mix by gently agitation of the media in the plate.

3. Co-transfect 5ug pME-VSV-G and 5 µg MFG-lacZ by the calcium phosphate precipitation method described above (section 3.4 Step 3).

4. Ten hours later, remove the media and replace with at least 5 ml of fresh GM.

5. Seventy two hours later, remove the retroviral supernatant which can either be used immediately, frozen, or concentrated (see step 6) (see Note ).

6. The viral supernatant can be concentrated with no apparent loss of titer by centrifugation at 25,000 RPM in an SW41 rotor for 3 hours or 50,000 x g at 5°C for 90 minutes to 2 hours.

7. Infect the target cells using 4 µg/ml Polybrene as described above.

Within 36 hours after transfection, syncytia can be noted forming in the transfected Anjou cell population. bgal staining of lacZ transfected Anjou cells after removal of viral supernatant accentuates visualization of syncytia formation.

Co-culturing of the target cells with producer cells is not recommended due to syncytia induction. We have also observed syncytia formation following infection of NIH3T3 target cells using concentrated or unconcentrated (base) virus supernatant. This is likely due to the carry-over of membrane fragments bearing VSV-G, which is known to be highly fusogenic.

Acknowledgments:

The authors are extremely grateful to David Baltimore for continued encouragement, advice, and support, and in whose laboratory many of the experiments were conceived and carried out. The authors would also like to thank their colleagues in the Baltimore lab, Nolan lab, and elsewhere for advice and suggestions regarding these protocols. W.S.P. is supported by a Howard Hughes Medical Institute Physician Postdoctoral Fellowship. G.P.N. is a Scholar of the Leukemia Society of America and a recipient of the Burrough's Wellcome New Investigator In Pharmacology Award. G.P.N. is supported by National Institutes of Health Grants NIHRO1AI35304 and a gift from Tularik, Inc.

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3. Watanabe, S. and Temin, H.M. (1983) Construction of a helper cell line for avian reticuloendotheliosis virus cloning vectors. Mol. Cell. Biol. 3, 2241-2249.

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