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NOTES Rapid Quantitation of Recombinant Retroviruses Jeffrey R. Morgan,SJ Jongwon Lee,? Ronald G. Tompkins,t and Martin L. YarmushtJ Surgical Services, Massachusetts General Hospital and the Shriners Burns Institute, Boston, Massachusetts 02114, and Department of Chemical and Biochemical Engineering, Rutgers University, Piscataway, New Jersey 08854
A method for measuring the concentration of recombinant retrovirus encoding the Escherichia coli LacZ gene is described. The assay is based on the quantitative measurement of 8-galactosidase activity in extracts of cells infected with the LacZencoding retrovirus. Lad-encoded 8-galactosidase activity in transduced cells is measured using the colorimetric substrate, o-nitrophenyl 8-D-galactopyranoside(ONPG), and the results are read using an ELISA plate-reader. Because the entire assay is performed in a 96-well plate, large numbers of samples are easily measured, and the assay has the advantages of rapidity, precision, and ease of data collection. The assay was used to determine the optimal concentration of polybrene, a polycation known to enhance the infectivity of retroviruses, and can be used to evaluate other factors that affect infection, as well as the optimal conditions for production of the recombinant retrovirus.
Introduction
to isolate continuously growing cell lines that constitutively
Recombinant retroviruses provide an efficient means for the introduction and expression of genes in mammalian cells (Varmus, 1988; Friedmann, 1989; Anderson, 1992; Mulligan, 1993). Retroviral mediated gene transfer has been widely used in research and has recently been approved for use in human studies protocols that seek to determine the utility of gene transfer for gene therapy (Friedmann, 1989; Anderson, 1992). Several human studies have been completed, and others are planned or in progress (Anderson, 1992). While results have been encouraging to date, technical issues related to retroviral mediated gene transfer still remain that need to be addressed before this technology is widely applied in the clinic (Mulligan, 1993). Specialized packaging cell lines have been developed for the production of recombinant retroviruses (Danos and Mulligan, 1988; Miller et al., 1986; Markowitz et al., 1988). These packaging cell lines are derivatives of 3T3 cells, into which the retroviral genes, gag, pol, and enu, have been introduced. The genomes encoding these retroviral genes have been suitably inactivated, so that the packaging cell lines are unable to produce wild-type replication-competent retroviruses, yet are capable of supplyingthe gene products necessary for the production of replication-defectiverecombinant retroviruses that can mediate gene transfer. When these packaging cell lines are transfected with DNA encoding a recombinant retrovirus, cell lines that produce infectious recombinant retroviral particles can be selected. The production of retroviral particles is nonlytic and occurs by budding at the cellmembrane(Varmus, 1988). Therefore, it is possible
* Address correspondence to Jeffrey R.Morgan, Ph.D., Bigelow 13, Trauma Services, Room 1302, Massachusetts General Hospital, Boston, MA 02114. Telephone: 617-726-3712. Fax: 617-726-4127. + Massachusetts GeneralHospitaland the Shriiers Burns Institute. t Rutgers University. 8756-7938/94/3010-0441$04.50/0
shed recombinant retroviral particles into the culture medium (Danos and Mulligan, 1988; Miller et al., 1986; Markowitz et al., 1988). Several methods have been used to determine the number of active recombinant retroviral particles (titer) produced by packaging cell lines. One of the first methods relied on the fact that the early retroviral vectors encoded, in addition to the gene of interest, a second gene encoding a dominant selectable marker, which conferred resistance to a drug or antibiotic (Miller and Rosman, 1989). After infection, the cells were plated into selective media containing the antibiotic; after 2 weeks the grown drugresistant colonies were stained and counted, and the titer of the stock was expressed as colony-forming units/ milliliter (cfu/mL). Other methods to determine the titer include Southern blot hybridization of DNA extracted from infected cells to determine how many gene copies have been transferred to the population of infected cells (Guild et al., 1988; Morgan and Eden, 1991) and fluorescence-activated cell sorting (FACS analysis) of infected cells to determine what proportion of an infected cell population expresses the new gene product (Strair et al., 1988; Sladek and Jacobberger, 1990). In this report, we describe a simple colorimetric method to measure the concentration of recombinant retroviruses encoding the LacZ gene (@-galactosidase).Following the infection of cells plated in a 96-well dish with stocks of LacZ retrovirus, cells are lysed and the amount of @-galactosidaseactivity in infected cells is quantitated using the substrate, o-nitrophenyl @-pgalactopyranoside (ONPG), and an ELISA plate-reader. The assay is rapid and quantitative and, thus, can be considered an advance over other methods used to determine the concentration of recombinant retrovirus. The LacZ gene has been used in many studies as a reporter gene and is useful for determining spatial as well as temporal patterns of gene expression. Cells expressing
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LacZ are easily identified by histological staining for 8-galactosidaseactivity in vitro, as well as in tissue sections (Sanes et al., 1986; Price et al., 1987). Thus, LacZ retroviruses injected into animal tissues have been used to label cells in vivo to map cell lineages during development and to determine the feasibility of direct in vivo gene transfer into tissues such as the liver (Kay et al., 1993). In addition, cells modified with the LacZ virus ex vivo and subsequently transplanted can be identified in tissue sections, and the fate of these transplanted cells can be easily determined (Wilson et al., 1989; Dhawan et al., 1991). The assay described in this report can be applied to the isolation and development of improved LacZ viruses with higher levels of expression, as well as higher viral titers. Using a LacZ virus as a model retrovirus in conjunction with this assay, which can handle large numbers of replicate samples, it should be possible to optimize the various steps of retroviral mediated gene transfer, and these results should be relevant to the use of other therapeutically important recombinant retroviruses.
Materials and Methods Chemicals. Nonidet P-40 (NP-40), o-nitrophenyl 8-Dgalactopyranoside (ONPG), glutaraldehyde (ultrapure), rhodamine B, and 1,5-dimethyl-l,5-diazaundecamethylene polymethobromide (polybrene) were purchased from Sigma Chemical Co. (St. Louis, MO). 5-Bromo-4-chloro3-indolyl fl-D-galactopyranoside (X-Gal) was purchased from Boehringer Mannheim GmbH (West Germany). Cell Culture. NIH 3T3's and Lac2 virus producing cell lines were cultured in Dulbecco's Modified Eagle's medium (DMEM) (Gibco BRL, Gaithersburg, MD) with 10% bovine calf serum (HyClone Labs Inc., Logan, UT) containing 100 units/mL penicillin and 100 pg/mL streptomycin (Gibco BRL). The amphotropic packaging cell line producing the a-SGC-Lac2 virus was kindly provided by L. Cohen of Somatix Therapy Corporation (Alameda, CA) (Dhawan et al., 1991). LacZ virus containing media were harvested from confluent cultures (overnight incubation of 10mL of culture medium in a 10-cmtissue culture dish) of the virus-producing cell line and then frozen of pulverized dry ice in 1-mL aliquots and stored at -85 "C. Diluted Titer Assay. LacZvirus titers were performed as previously described (Price et al., 1987). Tenfold serial dilutions of the frozen LacZ virus stock in culture media were adjusted to 8 pg/mL polybrene. Three milliliters of these dilutions were usedto infect triplicate 60-mm dishes, into which 2.6 X 106 3T3 cells had been plated the day before infection. Two days after infection, the media were removed, and the plates were washed once with PBS and fixed in PBS containing 0.5% glutaraldehyde for 5 min at room temperature. Plates were washed with PBS and 1mM MgCl2 and stained for Lac2 activity by incubation in a solution of PBS, 1 mM MgC12, 3.3 mM K4Fe(CN)s.3H20,3.3 mM K*e(CN)G, and 1 mg/mL 5-bromo4-chloro-3-indolyl~-~galactopyranoside (X-Gal) for 3-4 h at 37 "C. The plates were washed with PBS and allowed to air-dry. Colonies of LacZ+ cells (typically clusters of 2,4, or 8blue cells) were counted with the aid of a dissecting microscope. A t appropriate dilutions of the LacZ virus, the clusters of blue cells were sufficiently spread over the 60-mm dish so that each cluster arose from a single infectious event. Values are the average of at least three plates and two different dilutions. The Lac2 stock used in this study contained (4 f 0.8) X 106 LacZ+ colonyforming units/mL. Kinetic Assay. The day before infection with the LacZ virus, a 10-cm dish of confluent 3T3's was washed with
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PBS containing 5 mM ethylenedinitrilotetraaceticacid (EDTA) and treated with 2 mL of 0.05% trypsin in PBS, 2% glucose, and 2.5 mM EDTA for 2-5 min a t 37 "C. The trypsinized cells were counted using a Coulter Counter Model ZM (Coulter Electronics, Hialeah, FL), and 5000 cells in 100pL of medium were plated per well in a 96-well flat-bottom tissue culture dish with a low evaporation lid (Costar Corp., Cambridge, MA). The next day (19-25 h), media were removed and dilutions of LacZvirus in culture medium were made 8 pg/mL polybrene and added to each (final volume, 100 pL/well). The media were removed two days later, and the wells were washed with 100 pL of phosphate-buffered saline (PBS) containing 1mM MgCl2. The wash solution was removed, and 100pL of lysis buffer (PBS, 1 mM MgC12, and 0.5% NP-40) with substrate (3 mM o-nitrophenyl fl-D-gdactopyranoside, ONPG) prewarmed to 37 "C was added to each well (Lim and Chae, 1989). The plate was immediately placed in a THERMOmax plate-reader (Molecular Devices, Menlo Park, CA). The chamber temperature was set for 37 "C, and the optical density at 420 nm minus nonspecific background at 650 nm of each well was determined every minute over a period of 2 h. The maximum rate of reaction (V-) was calculated using the,,V points function of the plate-reader's software (SOFTmax). Vm, points are the number of points (25) used in a series of regressions to calculate the maximum rate. The Vm, values are reported in mOD/min (millioptical density units per minute) and are the average of triplicate wells. End-Point Assay. The conditions for viral infection for the end-point assay were the same as above. Two days after infection, the culture medium was removed and the cells were washed once with 100 pL of PBS and 1 mM MgCl2. After removal of the wash solution, lysis buffer was added (50pL) to each well, and the plate was incubated a t 37 "C. After 30 min, lysis buffer with ONPG prewarmed to 37 "C (PBS, 1 mM MgCl2, 0.5% NP-40, and 6 mM ONPG) was added (50pL) to each well, and the plate was incubated at 37 "C for another 15 min. The reactions were halted by the addition of 20 pL of stop buffer (1M Na2C03) (Sambrook et al., 1989). The plate was brought to room temperature, and the optical density was read at 420 nm minus nonspecific background at 650 nm. Values for each point are the average of triplicate wells. Clonal Assay. Confluent wells of the 96-welldish were washed once with PBS and 5 mM EDTA and treated with 100 FL of trypsin solution as described above. Cells were dispersed by repeated pipeting, and 20 pL of the trypsinized cells was diluted into 1mL of medium. Ten microliters of this dilution was plated into a 10-cm dish. After 10 days, when approximately 100 macroscopic colonies had grown, the plates were washed once with PBS and then fixed in PBS containing 0.5% glutaraldehyde for 5 min at room temperature. Plates were washed with PBS and 1mM MgC4 and stained for LacZ activity by incubation in a solution of PBS, 1 mM MgC12, 3.3 mM &Fe(CN)r3H20, 3.3 mM KSFe(CN)s, and 1 mg/mL 5-bromo-4-chloro-3-indolyl8-~galactopyranoside (X-Gal) for 3-4 h at 37 "C. Reaction mixes were removed, and the colonies were counterstained with a solution of 1% rhodamine B, rinsed with water, and air-dried. Values for the percentage of LacZ+ colonies a t each point (blue colonies/total colonies X 100) are the average of triplicate wells of the 96-well dish.
Results Dose Response of La&+ Cells. As a first step toward developing an assay for the measurement of recombinant
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Figure 1. Kinetics of 8-galactosidaseactivity in a single well. The increase in optical density is shownat 420 nm minus 650 nm for a single well containing LacZ+ 3T3 cells. Lysis buffer with ONPG was added to the well, and readings were performed every minute for 2 h while the temperature was maintained at 37 O C . retrovirus, we tested a cell l i e that constitutively expresses 0-galactosidase,the product of the E. coli LacZgene. This LacZ+ cell line is a clone of NIH 3T3 cells that has been transduced with the pLJ-Lac2 recombinant retrovirus (Price et al., 1987). LacZ+ cells were serially diluted in triplicate in a 96well plate to measure j3-galactosidase activity. Total cell numbers in each well were kept constant at 50 OOO cells/ well by the addition of normal NIH 3T3 cells. After the cells had attached, medium was removed, and the cells were washed with PBS and 1 mM MgC12 (washing solution). The cells were lysed with a solution of PBS, 1 mM MgC12, and 0.5% NP-40 (lysis buffer) containing 3 mM colorimetric substrate, o-nitrophenyl 8-D-galactopyranoside (ONPG). Immediately after the addition of ONPG, the optical density at 420 nm minus nonspecific background a t 650 nm was read each minute for 2 h at 37 "C in a THERMOmax kinetic plate-reader. The &galactosidase activity as a function of time as given by the plate-reader for a single well is shown in Figure 1. Analysis of these data appears to yield three distinct reaction phases: (1)a lag time of between 5 and 10 min, which varies between replicate wells; (2) a linear portion of the curve, where the rate is maximal; and (3) a later phase in which the reaction slows. The maximal velocity (V-) of each reaction in mOD/min was calculated from the slope of the line (25 points). A plot of V,, values for different dilutions of La&+ cells is shown in Figure 2. These results demonstrate that, between 780 and 50 000 L a d + cells/well, V,, values are directly proportional to the number of LacZ+ cells. Dose Response of Lac2 Virus. To adapt the assay to measure stocks of LacZ recombinant retrovirus, 5000 normal NIH 3T3 cells were plated per well, and medium containing the LacZ virus was added to each well the next day. After 2 days (sufficient time for the integration and expression of the LacZ recombinant retrovirus), the cells, which had grown to confluence in each well, were washed, lysed, and assayed for &galactosidase, as described above. A plot of V,, values for different dilutions of LacZ recombinantretrovirus is shown in Figure 3. These results show that V,, values are directly proportional to the volume of LacZ virus added per well over almost the entire range tested, from 6 to 50 pL/well. In addition, these results indicate that the assay is capable of detecting small volumes of LacZ virus (e.g., 6 pL). At virus volumes above 50 pL, the dose response curve appears to reach a plateau and may represent saturation of the infection of cells or a maximum for the production of &galactosidase by infected cells. The V,, for cells infected with undiluted viral stock (100 pL) was 25.7 mOD/min.
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Figure 3. (A, top) Diagram of the LacZrecombinant retrovirus. Shown are the retroviral long terminal repeats (LTR) and the Lac2 gene expressed from an internal enhancer/promoter combination, a-globin promoter (a),the extended packaging sequence (J.+), and cytomegalovirus enhancer (CMV). (B, bottom) Dose response curve for Lac2 recombinant retrovirus. Normal NIH 3T3 cells (5000/well) were plated and infected the next day with 100 pL of medium containing varying amounts of Lac2 virus with 4 pg/mL polybrene. Two days later, the cells were washed and lysed,and the.,V was determined. Each point shows the mean and standard deviation of the .,V for three replicates. Parallel wells of cells infected with the same virus dilutions were treated with trypsin, and the trypsinized cells were diluted and plated onto a 10-cm dish. After 10 days of growth,macroscopiccolonieswere fiied, stained forLac2activity using the X-Gal substrate, and counterstained with rhodamine B. Values for the percentage of LacZ+ colonies at each point (blue colonies/total colonies X 100)are the average and standard deviation of triplicate wells of the %-well dish. Clonal Assay. As an independent measurement of the dose response of LacZvirus, we determined the percentage of L a d + cells after infection. Instead of the measurement of the total enzymatic activity of a cellular extract, cells
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Figure 4. (A) Kinetics of release of &galactosidase after the addition of lysis buffer. LacZ+ NIH 3T3 cells (50 OOO cells/well) were plated and allowed to attach (4h), and lysis buffer without ONPG (50 pL) was added to each well. A t various times after the addition of lysisbuffer (0,3,5,15, and 30 min),the supernatantswere removed (40pL), and the V-values for &galactosidase in the supernatants and the wells were measured. Each point shows the mean and standard deviation of the V- for three re licates. (B) Relationship between lysis time and lag time. L a d + cells (50 OOO cells/well) were plated and allowed to attach (4 h), and L i s buffer without ONF'G (50 p L ) was added to each well. At various times after the addition of lysis buffer, ONPG in lysis buffer (50 pL) was added and the kinetics of 8-galactosidase activity was determined. The lag time wae measured from the kinetic curves for each well. Each point shows the mean and standard deviation of the lag time for three replicates. (C) Stability of &galactosidase in lysis buffer. L a d + NIH 3T3 cells (50 OOO celldwell) were plated and allowed to attach (4h), and lysis buffer without ONPG (50 pL) was added to each well. At various times after the addition of lysis buffer, ONPG in lysis buffer (50 pL) was added, and the V, values for B-galactosidase were determined. Each point shows the mean and standard deviation for three replicates. were plated at clonal density and allowed to grow into colonies, and the colonieswere stained for &galactosidase. Parallel wells of cells infected with the same Lac2 virus dilutions were treated with trypsin, diluted, and plated onto 10-cm dishes. After 10 days, when the colonies had grown to macroscopic size, the cells were fixed and stained for &galactosidase activity by incubation with the substrate X-Gal, which forms a blue precipitate in cells expressing the LacZ gene. Cells were counterstained with rhodamine B to reveal colonies of uninfected cells, and the percentages of LacZ+ colonies were counted as shown in Figure 3. A similar dose response curve for the Lac2 virus is seen when the percentage of LacZ+ colonies was determined using X-Gal staining. These results are in good agreement with those from the plate-reader assay. End-Point Assay. While these results were very encouraging, we sought to simplify the assay even further by reducing the measurement time of each plate. Therefore, we chose to develop an end-point assay in which the optical density is read after the reactions are stopped, thus increasing the versatility of the assay by allowing the reading of multiple plates within a short period of time. For an end-point assay to be accurate, the assay must be terminated in the linear phase of the reaction, and since all reactions are stopped after the same time interval, variations in the lag phase as shown in Figure 1 could adversely affect the accuracy of the assay. Therefore, we chose to investigate the early phase of the reaction and the contribution of early events (e.g., cell lysis) to the lag phase. The kinetics of release of 8-galactosidase after addition of the lysis buffer was examined by plating 50 000LacZ+ cells/well,allowing the cells to attach, and at various times after the addition of lysis buffer removing the supernatants and measuring &galactosidase activity. Likewise, 8-galactosidase remaining in the wells at various times after the addition of lysis buffer was measured by replacing the supernatants with additional lysis buffer containing ONPG. Figure 4A shows the kinetics of release and retention of &galactosidase after lysis. Maximal release of 8-galactosidase from lysed cells into the supernatant required lysis for a minimum of 5 min. Conversely, &galactosidase associated with the cells decreased to nearminimal levels 5 min after the addition of lysis buffer. The relationship between lysis time and lag time was examined further by adding lysis buffer to a series of wells containing 50 000 LacZ+ cells/well, followed by the
addition of ONPG a t various times. The lag time for each reaction was measured. Figure 4B shows that the length of the lag time steadily declined to zero as the time of ONPG addition was delayed. The lag time was negligible when ONPG was added at least 15 min after the addition of lysis buffer. The stability of the @-galactosidaseenzymein lysis buffer was also measured. LacZ+ cells (50 000 cells/well) were plated in a 96-well dish. The cells were lysed, and @-galactosidasewas measured a t different times after lysis. As shown in Figure 4C,8-galactosidase activity was stable in the lysis buffer, and V- levels remained unchanged for as long as 120 min after lysis. On the basis of the stability of &galactosidase in lysis buffer and the time needed for equilibration after lysis, we devised the following format for an end-point assay that effectivelyeliminates the lagphase and any associated variability. Lysis buffer without ONPG was added to the wells, and the plates were incubated at 37 "C. Lysis buffer with ONPG equilibrated to 37 "C was added after 30 min, and the reactions were incubated at 37 "C.The reactions were stopped after 15 min by the addition of sodium bicarbonate, and the optical density was then determined. To demonstrate the utility of the assay, the end-point assay was used to determine the optimal concentration of polybrene, a polycation commonly used to enhance the infectivity of cells by retroviruses. One day after 5000 NIH 3T3 cells/well were plated, a LacZ virus stock with varying concentrations of polybrene was added. After 2 days, &galactosidase activity was measured using the conditions for the end-point assay described above. Figure 5 shows that polybrene significantly increases infectivity when concentrations of polybrene between 4 and 16 pgl mL are used. At concentrations higher than 32 pgImL, infectivity was inhibited. Identical results were obtained when the kinetic assay (V-) was used to determine the optimal polybrene concentration (data not shown).
Discussion This report describes a rapid and quantitative assay for the determination of the concentration of recombinant retrovirus encoding the Lac2 gene. The method used is a colorimetric assay that uses the substrate, ONPG, and can be read using an ELISA plate-reader, which facilitates the handling of large numbers of samples and replicates. In addition, the software packages available with most
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PoLYBRENE (ug/m/l Figure 5. Optimal concentration of polybrene determined by the end-point assay. Normal NIH 3T3 cells (5000/well) were plated and infected the next day with 100 pL of medium containing LacZ virus with varying concentrationsof polybrene. Two days later, the end-point assay was performed. Cells were washed with 100 pL of PBS and 1 mM MgC12, lysed with 50 pL oflysisbufferfor30minat37 OC,andincubatedwithanadditional 50 p L of lysis buffer containing 6 mM ONPG at 37 O C . After 15 min, the reactions were stopped by the addition of 20 pL of 1M Na2C03. Optical density was read at 420 nm minus 650 nm. Each point shows the mean and standard deviation for three replicates. ELISA plate-readers facilitate the rapid handling and analysis of data. The concentration of LacZ virus can be determined in as little as 2 days after infection using this assay. In contrast, previous methods that measure the titer of recombinant retroviruses by counting drug-resistant colonies require as long as 2 weeks for the colonies to grow (Miller andRosman, 1989). TheLacZgenemaybeauseful alternative to the dominant selectable marker genes found in many current vectors, and this assay could be used in lieu of the drug-resistant colony-forming assay to quickly determine the concentration of the Lad-encoding recombinant retroviruses. Unlike assays that measure virus activity after serial dilution, this assay measures viral infectivity at relatively high concentrations (Le., undiluted stocks), which are typical of the conditions used for ex vivo gene therapy. In addition, the assay could bemodified to measure other recombinant retroviruses whose gene products can be quantitated with a colorometric assay (Schreiber et al., 1993). This assay measures virus concentration by measuring @-galactosidaseactivity. @-Galactosidaseactivity is proportional to both the number of LacZ+ cells and the level of LacZ expression in eachcell. Therefore, standard curves must be prepared for each Lac2 virus. Viruses with different promoters, and thus different levels of Lac2 expression, may not be comparable unless it is shown that they express equivalent levels of @-galactosidaseor that the expression levels can be normalized between viruses. A series of recombinant viruses expressing LacZ from the same internal promoter might be expected to produce comparable levels of @-galactosidase. Using a single LacZ virus as a model recombinant retrovirus, this assay should be useful for optimization of the major steps of retroviral mediated gene transfer. For example, the determination of the optimal conditions for adsorption and infection of the Lac2 (genetransfer) should be relevant to other amphotropic recombinant retroviruses. In this report, we have demonstrated one such application of this assay, namely, the determination of the optimal
concentration of polybrene, a polycation commonly used to enhance the infectivity of cells by retroviruses. Other reports have shown that growth factors can be used to enhance the infectivity of cells (Lim et al., 1989;Miller et al., 1990). The assay described in this report could be used to screen for other growth factorslagentslconditions that enhance the infectivity of those cell types considered targets for gene therapy and retroviral mediated gene transfer. We suspect that the assay could be easily modified for nonadherent cell types, such as lymphocytes, simply by including a centrifugation step prior to the addition of the wash and lysis solutions. Similarly, this assay, in conjunction with the LacZ virus, could be used to investigate the conditions governing the optimal production of recombinant retroviruses by packaging cell lines. Issues to be addressed in the future include the density of the packaging cell line and the time to harvest a virus, as well as factors that enhance production of recombinant retroviruses.
Acknowledgment This work was supported by NIH Grant PO1 HD2852801 (J.R.M.). Literature Cited Anderson, W. F. Human gene therapy. Science 1992,256,80813.
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Morgan, J. R.; Eden, C. A. Retroviral-mediated gene transfer into transplantable human epidermal cells. In Clinical and Experimental Approaches to Dermal and Epidermal Repair: Normal and Chronic Wounds, Progress in Clinical and Biological Research, Vol. 365; Barbul, A., Caldwell, M. D., Eaglstein, W. H., Eds.; Wiley-Liss: New York, 1991; pp 417428.
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Accepted March 7,1994.' Abstract published in Advance ACS Abstracts, May 1, 1994.
