Previous Article | Next Article 
Journal of Virology, October 1999, p. 8837-8842, Vol. 73, No. 10
Department of Genetics and Developmental
Biology,1 Mary Babb Randolph Cancer
Center,2 and Department of
Biochemistry,3 West Virginia University,
Morgantown, West Virginia 26506
Received 18 March 1999/Accepted 6 July 1999
Expression of the selectable drug resistance gene in retroviral
vectors used for gene therapy can lead to a decreased expression of the
gene of interest and may induce a host immune response, resulting in a
decreased efficiency of gene therapy. In this study, we demonstrate
that high-frequency deletion of direct repeats, an inherent property of
reverse transcriptases, can be used to efficiently excise the drug
resistance gene during reverse transcription. One retroviral vector
containing a direct repeat deleted the neomycin resistance expression
cassette during a single replication cycle at >99% efficiency.
Most retroviral vectors used for
gene therapy are based on murine leukemia virus (MLV) and express a
gene of interest as well as a drug resistance gene. A drug resistance
gene is typically necessary for selection of virus-producing helper
cells or cell clones so that high virus titers can be achieved. The
bacterial neomycin phosphotransferase gene (neo) is commonly
used as a selectable marker in retroviral vectors (35). The
expression of two genes can be achieved by using the viral long
terminal repeat (LTR) promoter to express the gene of interest and an
internal promoter to express the drug resistance gene. Expression of
the gene of interest and the drug resistance gene from different
promoters often results in promoter interference and reduction of the
expression of either gene in comparison to the levels of expression
from vectors bearing only one gene (1, 4). These differences have been attributed to promoter interference between the 5' LTR promoter and the internal promoter and to the downregulation of the LTR
promoter in certain cell types (1, 2, 4, 11, 12, 32).
Usually, the nonselected gene of interest is expressed at a level lower
than that of the selected drug resistance gene. Alternatively, an
internal ribosomal entry site (IRES) can be used instead of an internal
promoter to express two gene products from one RNA transcript
(18-20). In one study, however, expression of the
IRES-neo cassette from a retroviral vector reduced the expression of the nonselected genes of interest (5).
Therefore, removal of the drug resistance gene along with its promoter
or translational control regions from the vector during infection may
lead to an enhanced and sustained expression of the gene of interest.
It is also desirable to remove the drug resistance gene from target
cells during gene therapy so that unnecessary expression of a foreign
protein can be avoided. Expression of the drug resistance gene in cells
can lead to development of a host immune response against the
transduced cells, which may reduce the long-term efficacy of gene
therapy (25, 33). Specific T-cell responses to components of
the retroviral vector, such as the product of the selectable marker
gene hygromycin phosphotransferase B, have been observed (22).
The Cre/loxP recombination system from bacteriophage P1 was
recently used to delete a neomycin resistance expression unit from
proviruses in transduced hematopoietic cells (13). In this system, it is necessary that the infected target cells express the Cre
recombinase protein and that the loxP sites flank the drug
resistance expression unit. The Cre recombinase can be expressed either
from the retroviral vector bearing the gene of interest or from a
separate expression vector. The effects of long-term expression of the
Cre recombinase in target cells on the stability of the human genome
are unclear. While this system may be applicable to ex vivo gene
therapy approaches, its usefulness in the deletion of the drug
resistance gene in the context of in vivo gene therapy may be limited
because of the need for expression of the Cre recombinase in the target
cells. Additionally, the frequency of deletion of the neo
resistance expression unit was observed to be 74% (13). It
was hypothesized that frequent rearrangements in the retroviral vectors
resulted in the loss of one or both loxP sites, which reduced the efficiency of excision.
Directly repeated sequences have been shown to be deleted accurately
and at high frequencies in both spleen necrosis virus- and MLV-based
retroviral vector systems (9, 21, 30). It has long been
observed that directly repeated sequences found within retroviral
genomes are unstable (8, 17, 28, 31, 36). Deletion of direct
repeats occurs during reverse transcription and involves the viral
reverse transcriptase dissociating from one copy of the direct repeat
and reassociating with the homologous sequence in the second copy of
the direct repeat (10, 21).
We previously used the high frequency of direct-repeat deletion to
develop self-inactivating and self-activating vectors based on both MLV
and spleen necrosis virus (9, 21). It was shown that
directly repeated sequences could be used to efficiently delete the
viral packaging signal and functionally reconstitute neo or
the herpes simplex virus thymidine kinase gene (HTK) during reverse
transcription. A 701-bp direct repeat composed of overlapping fragments
of HTK was deleted at a rate of 57% and functionally reconstituted HTK
in one replication cycle (9). When the same direct repeat
flanked the MLV encapsidation sequence ( In this study, we sought to establish whether direct repeats could be
used to efficiently delete drug resistance genes and their control
regions. Previous studies indicated that direct repeats could be used
to delete at least 818 bp of viral sequence (length of Construction of MLV-based retroviral vectors.
To determine the
efficiency of using direct repeats for deletion of selectable markers,
the vectors pKD-HTneoTK and pKD-HT Protocol to determine the deletion frequency of KD-HTneoTK and
KD-HT Frequency of direct-repeat deletion determined by viral titers
obtained from pools of G418-resistant packaging cells.
The results
of infections with viruses derived from pools of G418-resistant
packaging cells are summarized in Table
1. The G418 titers represent the
population of viruses that did not undergo a direct-repeat deletion,
whereas the HAT titers represent the population of viruses that
underwent direct-repeat deletion and reconstituted a functional HTK.
Therefore, the frequency of direct-repeat deletion and reconstitution
of HTK was determined by dividing the HAT titers by the sum of the G418
and HAT titers. KD-HTneoTK and KD-HT
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Development of Murine Leukemia Virus-Based
Self-Activating Vectors That Efficiently Delete the Selectable Drug
Resistance Gene during Reverse Transcription
ABSTRACT
Top
Abstract
Text
References
TEXT
Top
Abstract
Text
References
), the deletion frequency
increased to 91%. The provirus in the infected target cells lacked and expressed a functional HTK.
) during
reverse transcription (9). Most of the drug resistance genes
and their control regions range in length from approximately 1.5 kb
(for example, the simian virus 40 promoter plus the hygromycin B
phosphotransferase gene [14]) to 4 kb (for example,
the simian virus 40 promoter plus the Na+-K+
ATPase gene that confers resistance to ouabain [23]).
The effect of increasing linear distance between direct repeats on the
frequency of deletion was unknown. Therefore, it was not clear whether
direct repeats could be used to delete longer sequences encoding drug resistance genes and their control regions. In this report, we demonstrate that direct repeats can be used to delete the
neo selectable marker and its translational control region
at >99% efficiency.
neoTK were constructed by standard
procedures (see Fig. 1A and 2A) (34). Viruses derived from
these vectors are named KD-HTneoTK and KD-HTneoTK, respectively. A
detailed description of all cloning steps is available upon request.
neoTK after one round of viral replication.
Vectors
pKD-HTneoTK and pKD-HTneoTK (10 µg each per 60-mm-diameter dish)
were transfected into PG13 helper cells by calcium phosphate
precipitation as previously described (34). PG13 cells were
subjected to G418 selection (an analog of neomycin) at a final
concentration of 600 µg/ml (0.87 mM; Gibco). Approximately 2,500 G418-resistant colonies were separately pooled and expanded from each
transfection. For each vector, virus was harvested from transfected
G418-resistant cells and used to infect 143B target cells plated at a
density of 2 × 105 cells per 60-mm-diameter dish in
the presence of Polybrene (50 µg/ml) as previously described
(16). The target 143B cells are a TK-deficient human
osteosarcoma cell line (obtained from the American Type Culture
Collection). Infected 143B cells were subjected to either G418 (400 µg/ml, 0.58 mM) or hypoxanthine-aminopterin-thymidine (HAT; as
specified by Boehringer Mannheim) selection 1 day after infection. Two
weeks later, drug-resistant colonies were counted and viral titers were
determined from results of four to eight independent experiments. All
cells were maintained in Dulbecco's modified Eagle's medium (ICN
Biomedicals) supplemented with penicillin (50 U/ml; Gibco),
streptomycin (50 µg/ml; Gibco), and bovine calf serum (10% for PG13
and 6% for 143B; Hyclone Laboratories).
neoTK underwent direct-repeat
deletion and excised the IRES-neo cassette with average
deletion frequencies of 91% ± 0.8% and 93% ± 1.8%, respectively.
Thus, the frequencies of deletion observed were consistent between
independent experiments. The high frequency of deletion was consistent
with our recent observation that increasing distance between direct
repeats generally increases the frequency of deletion (10).
TABLE 1.
Virus titers after infection with KD-HTneoTK or
KD-HT neoTK pools
Structures of deleted and undeleted KD-HTneoTK proviruses
determined by Southern blot analysis.
Genomic DNAs were isolated
from pools of infected 143B cells that were selected for either G418 or
HAT resistance and analyzed by Southern hybridization by standard
procedures (34). The pools of cells were derived from
infections with low dilutions of virus (infected with undiluted or
10-fold-diluted virus) and contained at least 2,000 G418- or
HAT-resistant 143B cell colonies. A 1.3-kb DNA fragment encoding the
HTK gene was used to generate a probe with [
-32P]dCTP
(specific activity, >109 cpm/µg; ICN Biomedicals) with a
Random Priming DNA-Labeling Kit (Boehringer Mannheim). The expected
structures of the deleted and undeleted proviruses derived from
KD-HTneoTK are shown in Fig. 1A. Results
of a representative Southern blot analysis of genomic DNAs derived from
pools of cells infected with KD-HTneoTK and digested with
XbaI are shown in Fig. 1B. The expected 5.0-kb undeleted
band was detected in genomic DNAs from two independent pools of
G418-resistant cells (G418 lanes A and B). The expected 2.9-kb deleted
band was also detected in genomic DNAs from two independent pools of
HAT-resistant cells (HAT lanes A and B). As expected, the 2.9-kb
deleted band was not detectable in G418-resistant cells and the 5.0-kb
undeleted band was not detectable in HAT-resistant cells. Faint
additional bands that ranged in size from 3 to 5 kb were detected in
G418-resistant cells (G418 lanes A and B). The process of transfection
is mutagenic, and these bands may have resulted from deletions and
rearrangements during transfection of the packaging cells
(6). The results of these experiments indicated that the
IRES-neo cassette was efficiently deleted from proviruses
that were selected for resistance to HAT. However, it was possible that
some of the HAT-resistant cells contained two proviruses, one
conferring resistance to HAT and the other conferring resistance to
G418. Based on the sensitivity of Southern blot analysis, <10% of the
HAT-resistant cells were infected with another virus that retained the
IRES-neo cassette.
|
Most KD-HTneoTK-infected cells that are resistant to HAT are
sensitive to G418.
To more accurately determine the frequency of
proviruses that retained the IRES-neo cassette in cells
selected for resistance to HAT, HAT-resistant cells were plated at low
densities and subjected to G418 selection (Table
2). HAT-resistant 143B cells obtained from infections with low dilutions of virus (undiluted and
10-fold-diluted virus; multiplicity of infection [MOI], <0.005) were
seeded at low densities of 20, 40, or 80 cells per 60-mm-diameter dish
(experiment 4 with KD-HTneoTK [Table 1]). Sixty dishes were plated
for each cell density, and 30 dishes each were subjected to either HAT or G418 selection. A total of 3,896 HAT-resistant colonies and 157 G418-resistant colonies were obtained from cells infected with low
dilutions of virus. The results indicated that approximately 4% of the
HAT-resistant cells were also resistant to G418. In addition,
HAT-resistant 143B cells obtained from infections with high dilutions
of virus (100- and 1,000-fold-diluted virus; MOI, <0.00005) were
seeded at a density of 10 cells per 60-mm-diameter dish. Sixty dishes
were plated, and 30 dishes each were subjected to either HAT or G418
selection. A total of 303 HAT-resistant colonies were obtained from
cells infected with high dilutions of virus. In contrast to the results
obtained with cells that were infected with low dilutions of virus,
G418-resistant colonies were undetectable in pools that were infected
with high dilutions of virus. Therefore, <0.3% (<1/303) of the
HAT-resistant cells that were infected with high dilutions of virus
were resistant to G418.
|
Structures of deleted and undeleted KD-HTneoTK proviruses
determined by Southern blot analysis.
Genomic DNAs were isolated
from pools of KD-HTneoTK-infected 143B cells that were selected for
either G418 or HAT resistance and analyzed by Southern hybridization
(34). Each pool of HAT-resistant cells contained at least
4,000 independent colonies. However, because of the low number of
G418-resistant colonies obtained, each pool of G418-resistant cells
contained approximately 300 to 400 independent colonies. The genomic
DNAs were digested with XbaI and analyzed by Southern blot
analysis. The expected structures of the deleted and undeleted
proviruses derived from KD-HTneoTK are shown in Fig.
2A. The results of a representative
Southern blot analysis of genomic DNAs derived from pools of cells
infected with KD-HTneoTK are shown in Fig. 2B. The expected 5.0-kb
undeleted band was detected in genomic DNAs from two pools of
G418-resistant cells (G418 lanes A and B). The expected 2.1-kb deleted
band was also detected in genomic DNAs from two pools of HAT-resistant cells (HAT lanes A and B). As expected, the 2.1-kb deleted band was not
detectable in G418-resistant cells and the 5.0-kb undeleted band was
not detectable in HAT-resistant cells. These results indicated that the
IRES-neo cassette was efficiently deleted from proviruses
that were selected for resistance to HAT. Based on the sensitivity of
Southern blot analysis, >90% of the proviruses had deleted the
IRES-neo cassette.
|
Analysis of PG13 helper cell clones containing KD-HTneoTK.
To determine whether the aberrant bands observed in the G418-resistant
pools were the result of mutagenic transfection or aberrant splicing,
single-cell helper clones of KD-HTneoTK were isolated. The vector,
pKD-HTneoTK, was transfected into PA317 helper cells, an amphotropic
MLV helper cell line (26). Virus from pools of
G418-resistant cells composed of greater than 600 colonies was
harvested and used to infect PG13 helper cells. Twelve G418-resistant
PG13 cell clones were isolated and screened by Southern blot analysis
to verify the vector structure by three different restriction enzyme
digestions (data not shown). Additionally, each helper cell clone was
shown to contain only one KD-HTneoTK provirus by Southern blot
analysis (data not shown). Only 3 of the 12 cell clones analyzed
contained an intact provirus, and the remaining 9 cell clones contained
proviruses that exhibited deletions and rearrangements.
Deletion frequency of KD-HTneoTK clones determined by viral
titers.
Virus was harvested from the three KD-HTneoTK PG13
clones containing an intact provirus and used to infect 143B cells as previously described. The infected 143B cells were subjected to either
G418 or HAT selection, and the virus titers were determined (Table
3). The high HAT-resistant titers and low
G418-resistant titers indicated that direct-repeat deletion and
reconstitution of a functional HTK occurred at a high rate. The
deletion frequencies ranged from 98 to >99%, indicating that the vast
majority of proviruses in target cells had deleted the
IRES-neo cassette. Therefore, helper cell clones provided a
higher frequency of deletion (99%) than pools of helper cells
(94%; P = 0.002, two-sample t test).
|
Deletion frequencies of KD-HTneoTK clones determined by Southern
blot analysis.
Pools of 143B cells infected with virus were
derived from two of the PG13 cell clones. At least 2,000 HAT-resistant
colonies and <400 G418-resistant colonies were separately pooled and
expanded. Genomic DNAs isolated from the pools were digested with
XbaI and analyzed by Southern blotting (Fig. 2C). Similar to
the results obtained from infections with pools of helper cells (Fig.
2B), the expected 5.0-kb undeleted band was detected in genomic DNAs from two pools of G418-resistant cells (Fig. 2C, G418 lanes 1 and 2).
The expected 2.1-kb deleted band was also detected in genomic DNAs from
two pools of HAT-resistant cells (HAT lanes 1 and 2). As expected, the
2.1-kb deleted band was not detectable in G418-resistant cells and the
5.0-kb undeleted band was not detectable in HAT-resistant cells.
neoTK provirus in the PG13 helper cell clones had the
expected structure. Furthermore, since a single intact provirus was
present in each cell clone, the additional bands resulted, at least
partially, from aberrant splicing of the viral RNA in the helper cell clones.
Further consideration of the structure of the KD-HTneoTK provirus
supports the notion that aberrant splicing is responsible for the
additional bands observed in pools of G418-resistant cells. During MLV
replication, a full-length mRNA used for expression of
gag-pol and a spliced mRNA used for expression of
env are generated (7). The full-length viral mRNA
is packaged into virions, but the spliced message is not packaged
because it lacks the . The MLV splice donor site that is used to
express the viral env is located just 5' to the HT fragment.
This splice donor site can utilize any cryptic splice acceptor sites
that may be present in the HT fragment to give rise to spliced RNAs.
These spliced RNAs are expected to contain and are therefore
expected to be efficiently packaged into virions. Identification of the
4.3- and 4.0-kb proviral bands in Fig. 2C (G418 lanes 1 and 2) suggests that spliced RNAs lacking approximately the first 700 and 1,000 bp of
HT, respectively, were generated. Furthermore, reverse transcription of
these spliced RNAs should result in proviruses that can confer resistance to G418 but not HAT, since a portion of the HT fragment is
deleted. Therefore, the resulting G418-resistant titers for KD-HTneoTK do not fully represent the population of viruses that did
not undergo direct-repeat deletion. This population also contains spliced RNAs that were unable to undergo direct-repeat deletion but
still retained resistance to G418. Thus, the frequency of direct-repeat
deletion for KD-HTneoTK is likely to be higher than indicated in
Table 3 (>99%).
In conclusion, self-activating and self-inactivating retroviral vectors
containing directly repeated sequences can be used to efficiently
delete drug resistance genes and their control regions from retroviral
vectors during the course of reverse transcription. Deletion of the
selectable marker gene and its control regions may be used to prevent
promoter interference and increase the expression of the therapeutic
gene. Deletion of the selectable marker may also decrease the
probability of eliciting an immune response against the therapeutically
infected cells.
The efficiency of deletion of IRES-neo (>99%) with direct
repeats was higher than the 74% efficiency observed with the
Cre/loxP system (13). The advantages of using
direct repeats for deletion of selectable markers are that it is not
necessary to express potentially harmful proteins in the target cells
or include loxP sequences in the vector, which are subject
to a high rate of mutation. Therefore, direct-repeat vectors can be
used to easily delete the selectable markers in the course of in vivo
gene therapy.
The directly repeated sequences may be derived from any source,
including the therapeutic gene that is being delivered to target cells.
We have observed that direct repeats derived from several different
sequences, including those of HTK, neo, bacterial 
-galactosidase, green fluorescent protein, and the M13
bacteriophage, delete at very high rates (references 3, 9, 15,
21, and 30 and data not shown). Therefore,
it is very likely that overlapping fragments derived from any gene of
interest can be used to delete the selectable marker genes from
retroviral vectors. This strategy can be used to ensure that the
provirus in the target cell will express only the gene of interest.
It should be noted that it is possible to generate high-titer stocks of
retroviral vectors lacking selectable markers by using highly
transfectable packaging cell lines and transient transfection (24,
27, 29, 37). Nevertheless, it may be desirable to generate helper
cell clones containing an integrated provirus. Since the process of
transfection is known to be mutagenic (6), it is possible
that a high proportion of the retroviral vectors produced from these
systems express mutated gene products. Therefore, construction of
stable helper cell clones could be used to verify the integrity of the
gene of interest before infection of target cells. In addition, stable
packaging cell clones may provide better quality control, since
variations between different transfections can be avoided.
Future studies will be aimed at improving the virus titers of these
vectors by selection of high-titer-producing cell clones, deletion of
the viral splice donor site, and insertion of the HT fragment at
different locations upstream of the . These manipulations should
improve the viral titers by at least 100-fold.
| |
ACKNOWLEDGMENTS |
|---|
We especially thank Wei-Shau Hu for her intellectual input throughout the project. We also thank Jeffrey Anderson, Ben Beasley, Sara Cheslock, Que Dang, Elias Halvas, Carey Hwang, and Wenhui Zhang for helpful comments on the manuscript and for valuable discussion of the results.
This work was supported by Public Health Service grant CA58875 from the National Institutes of Health and American Cancer Society grant VM84706 to V.K.P.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, WV 26506. Phone: (304) 293-0495. Fax: (304) 293-4667. E-mail: VPATHAK{at}wvu.edu.
| |
REFERENCES |
|---|
|
Top
Abstract
Text
References
|
|---|
| 1. |
Apperley, J. F.,
B. D. Luskey, and D. A. Williams.
1991.
Retroviral gene transfer of human adenosine deaminase in murine hematopoietic cells: effect of selectable marker sequences on long-term expression.
Blood
78:310-317 |
| 2. | Artelt, P., R. Grannemann, C. Stocking, J. Friel, J. Bartsch, and H. Hauser. 1991. The prokaryotic neomycin-resistance-encoding gene acts as a transcriptional silencer in eukaryotic cells. Gene 99:249-254[Medline]. |
| 3. |
Bowman, R. R.,
W. S. Hu, and V. K. Pathak.
1998.
Relative rates of retroviral reverse transcriptase template switching during RNA- and DNA-dependent DNA synthesis.
J. Virol.
72:5198-5206 |
| 4. |
Bowtell, D. D. L.,
S. Cory,
G. R. Johnson, and T. J. Gonda.
1988.
Comparison of expression in hemopoietic cells by retroviral vectors carrying two genes.
J. Virol.
62:2464-2473 |
| 5. | Byun, J., J. M. Kim, P. D. Robbins, and S. Kim. 1998. The selectable marker neo gene down-regulates gene expression from retroviral vectors containing an internal ribosome entry site. Gene Ther. 5:1441-1444[Medline]. |
| 6. |
Calos, M. P.,
J. S. Lebkowski, and M. R. Botchan.
1983.
High mutation frequency in DNA transfected into mammalian cells.
Proc. Natl. Acad. Sci. USA
80:3015-3019 |
| 7. | Coffin, J. M. F. 1996. Retroviridae: the viruses and their replication. In B. N. Fields, D. M. Knipe, and P. M. Howley (ed.), Fields virology, vol. 2. Lippincott-Raven Publishers, Philadelphia, Pa. |
| 8. | Czernilofsky, A. P., A. D. Levinson, H. E. Varmus, J. M. Bishop, E. Tischer, and H. M. Goodman. 1980. Nucleotide sequence of an avian sarcoma virus oncogene (src) and proposed amino acid sequence for gene product. Nature 287:198-203[Medline]. |
| 9. |
Delviks, K. A.,
W. S. Hu, and V. K. Pathak.
1997.
 vectors: murine leukemia virus-based self-inactivating and self-activating retroviral vectors.
J. Virol.
71:6218-6224[Abstract].
|
| 10. |
Delviks, K. A., and V. K. Pathak.
1999.
Effect of distance between homologous sequences and 3' homology on the frequency of retroviral reverse transcriptase template switching.
J. Virol.
73:7923-7932 |
| 11. | Emerman, M., and H. M. Temin. 1984. Genes with promoters in retrovirus vectors can be independently suppressed by an epigenetic mechanism. Cell 39:449-467[Medline]. |
| 12. |
Emerman, M., and H. M. Temin.
1986.
Quantitative analysis of gene suppression in integrated retrovirus vectors.
Mol. Cell. Biol.
6:792-800 |
| 13. | Fernex, C., P. Dubreuil, P. Mannoni, and C. Bagnis. 1997. Cre/loxP-mediated excision of a neomycin resistance expression unit from an integrated retroviral vector increases long terminal repeat-driven transcription in human hematopoietic cells. J. Virol. 71:7533-7540[Abstract]. |
| 14. | Gritz, L., and J. Davies. 1983. Plasmid-encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae. Gene 25:179-188[Medline]. |
| 15. | Hu, W. S., E. H. Bowman, K. A. Delviks, and V. K. Pathak. 1997. Homologous recombination occurs in a distinct retroviral subpopulation and exhibits high negative interference. J. Virol. 71:6028-6036[Abstract]. |
| 16. |
Hu, W. S., and H. M. Temin.
1990.
Genetic consequences of packaging two RNA genomes in one retroviral particle: pseudodiploidy and high rate of genetic recombination.
Proc. Natl. Acad. Sci. USA
87:1556-1560 |
| 17. | Hughes, S., and E. Kosik. 1984. Mutagenesis of the region between env and src of the SR-A strain of Rous sarcoma virus for the purpose of constructing helper-independent vectors. Virology 136:89-99[Medline]. |
| 18. |
Jang, S. K.,
M. V. Davies,
R. J. Kaufman, and E. Wimmer.
1989.
Initiation of protein synthesis by internal entry of ribosomes into the 5' nontranslated region of encephalomyocarditis virus RNA in vivo.
J. Virol.
63:1651-1660 |
| 19. |
Jang, S. K.,
H. G. Krausslich,
M. J. Nicklin,
G. M. Duke,
A. C. Palmenberg, and E. Wimmer.
1988.
A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation.
J. Virol.
62:2636-2643 |
| 20. | Jorgensen, R. A., S. J. Rothstein, and W. S. Reznikoff. 1979. A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. Mol. Gen. Genet. 177:65-72[Medline]. |
| 21. |
Julias, J. G.,
D. Hash, and V. K. Pathak.
1995.
E vectors: development of novel self-inactivating and self-activating retroviral vectors for safer gene therapy.
J. Virol.
69:6839-6846[Abstract].
|
| 22. | Jung, D., E. Jaeger, S. Cayeux, T. Blankenstein, C. Hilmes, J. Karbach, U. Moebius, A. Knuth, C. Huber, and B. Seliger. 1998. Strong immunogenic potential of a B7 retroviral expression vector: generation of HLA-B7-restricted CTL response against selectable marker genes. Hum. Gene Ther. 9:53-62[Medline]. |
| 23. |
Kent, R. B.,
J. R. Emanuel,
Y. Ben Neriah,
R. Levenson, and D. E. Housman.
1987.
Ouabain resistance conferred by expression of the cDNA for a murine Na+, K+-ATPase alpha subunit.
Science
237:901-903 |
| 24. | Kinsella, T. M., and G. P. Nolan. 1996. Episomal vectors rapidly and stably produce high-titer recombinant retrovirus. Hum. Gene Ther. 7:1405-1413[Medline]. |
| 25. | McCormack, J. E., D. Martineau, N. DePolo, S. Maifert, L. Akbarian, K. Townsend, W. Lee, M. Irwin, N. Sajjadi, D. J. Jolly, and J. Warner. 1997. Anti-vector immunoglobulin induced by retroviral vectors. Hum. Gene Ther. 8:1263-1273[Medline]. |
| 26. |
Miller, A. D., and C. Buttimore.
1986.
Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production.
Mol. Cell. Biol.
6:2895-2902 |
| 27. | Murdoch, B., D. S. Pereira, X. Wu, J. E. Dick, and J. Ellis. 1997. A rapid screening procedure for the identification of high-titer retrovirus packaging clones. Gene Ther. 4:744-749[Medline]. |
| 28. |
Omer, C. A.,
K. Pogue-Geile,
R. Guntaka,
K. A. Staskus, and A. J. Faras.
1983.
Involvement of directly repeated sequences in the generation of deletions of the avian sarcoma virus src gene.
J. Virol.
47:380-382 |
| 29. | Onodera, M., A. Yachie, D. M. Nelson, H. Welchlin, R. A. Morgan, and R. M. Blaese. 1997. A simple and reliable method for screening retroviral producer clones without selectable markers. Hum. Gene Ther. 8:1189-1194[Medline]. |
| 30. |
Pathak, V. K., and H. M. Temin.
1990.
Broad spectrum of in vivo forward mutations, hypermutations, and mutational hotspots in a retroviral shuttle vector after a single replication cycle: deletions and deletions with insertions.
Proc. Natl. Acad. Sci. USA
87:6024-6028 |
| 31. |
Rhode, B. W.,
M. Emerman, and H. M. Temin.
1987.
Instability of large direct repeats in retrovirus vectors.
J. Virol.
61:925-927 |
| 32. | Richards, C. A., and B. E. Huber. 1993. Generation of a transgenic model for retrovirus-mediated gene therapy for hepatocellular carcinoma is thwarted by the lack of transgene expression. Hum. Gene Ther. 4:143-150[Medline]. |
| 33. | Riddell, S. R., M. Elliott, D. A. Lewinsohn, M. J. Gilbert, L. Wilson, S. A. Manley, S. D. Lupton, R. W. Overell, T. C. Reynolds, L. Corey, and P. D. Greenberg. 1996. T-cell mediated rejection of gene-modified HIV-specific cytotoxic T lymphocytes in HIV-infected patients. Nat. Med. 2:216-223[Medline]. |
| 34. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 35. | Southern, P. J., and P. Berg. 1982. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J. Mol. Appl. Genet. 1:327-341[Medline]. |
| 36. |
Tikhonenko, A. T., and M. L. Linial.
1993.
Transforming variants of the avian myc-containing retrovirus FH3 arise prior to phenotypic selection.
J. Virol.
67:3635-3638 |
| 37. | Yang, S., R. Delgado, S. R. King, C. Woffendin, C. S. Barker, Z. Y. Yang, L. Xu, G. P. Nolan, and G. J. Nabel. 1999. Generation of retroviral vector for clinical studies using transient transfection. Hum. Gene Ther. 10:123-132[Medline]. |
Journal of Virology, October 1999, p. 8837-8842, Vol. 73, No. 10
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Hu, W.-S., Pathak, V. K.
(2000). Design of Retroviral Vectors and Helper Cells for Gene Therapy. Pharmacol. Rev.
52: 493-512
[Abstract] [Full Text] -
Cheslock, S. R., Anderson, J. A., Hwang, C. K., Pathak, V. K., Hu, W.-S.
(2000). Utilization of Nonviral Sequences for Minus-Strand DNA Transfer and Gene Reconstitution during Retroviral Replication. J. Virol.
74: 9571-9579
[Abstract] [Full Text] -
Delviks, K. A., Pathak, V. K.
(1999). Effect of Distance between Homologous Sequences and 3' Homology on the Frequency of Retroviral Reverse Transcriptase Template Switching. J. Virol.
73: 7923-7932
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»