VOL. 6. 1986 
RETROVIRUS PACKAGING CELL LINES 
with the exception of PA12 cells containing the Neo virus 
pN2. Neither have we observed helper virus production 
from Psi-2 cells, except after introduction of the N2 vector. 
However, we know of two examples from other laboratories 
in which helper virus arose in PA12 cells in the absence of 
any apparent source of contamination. We confirmed the 
presence of helper virus in one case (unpublished data). 
Perhaps at very low frequency helper virus production can 
occur, possibly as a result of recombination of the packaging 
system with endogenous retrovirus like DNA or RNA ele- 
ments found in mouse cells. This phenomenon should be 
significantly reduced in the new packaging cell lines de- 
scribed here. 
The Neo virus N2 interacted with PA12 or Psi-2 packaging 
cells to yield helper virus at high frequency. We hypothesize 
that this is due to copackaging of packaging system RNA and 
Neo virus RNA, followed by recombination in the common 
gag region during reverse trancription in an infected cell. 
This event occurs in packaging cell populations, as clonal 
tines can be isolated that are initially helper free but produce 
helper virus eventually. Whereas cells infected with replica- 
tion-competent virus are resistant to reinfection with virions 
having the same pseudotype, PA12 and Psi-2 cells are much 
less resistant (16); thus, infection and recombination be- 
tween copackaged RNAs can occur in these packaging cell 
lines. In contrast, we did not detect helper virus production 
from PA317 cells containing N2 virus. A double recombina- 
tion event would be required between the packaging system 
and N2 virus in this case, which must be rare. N2 virus has 
characteristics which make it a useful vector. For instance, 
in canine and human marrow infections, only N2 and an- 
other virus containing gag sequences permitted efficient 
infection (9, 11a). The PA317 cell line now permits produc- 
tion of N2 virus in the absence of helper virus, and this utility 
probably extends to other potentially useful vectors which 
interact with previously available packaging cell lines to 
produce helper virus. 
Retroviral vectors produced by PA317 cells could infect 
mouse, rat, cat, dog, and human cells (data not shown), and 
thus they have an amphotropic host range. In particular, we 
were able to infect hemopoietic progenitor cells from human 
bone marrow using retroviral vectors secreted from PA317 
cells (R. A. Hock and A. D. Miller, unpublished data). 
Vector titer from the cells was also very high (up to 10 7 
CFU/ml); thus, these cells should be useful in experiments 
aimed toward human gene therapy. 
ACKNOWLEDGMENTS 
•We thank Eli Gflboa and David Trauber for supplying retroviral 
plasmids pN2 and pSDHT, respectively, Ming -Fan Law and Inder 
Verma for help in the early stages of these experiments, and Paul 
Neiman and Maxine Linial for comments on the manuscript. 
A.D.M. is a Special FeDow of the Leukemia Society of America. 
This study was supported by a grant from Cooley's Anemia Foun- 
dation and Public Health Service grant CA41455 awarded by the 
National Cancer Institute. 
LITERATURE CITED 
1. Anderson, W. F. 1984. Prospects for human gene therapy. 
Science 226:401-409. 
2. Colbere-Garaptn, F., S. Cbousterman, F. Horudniceans, P. 
Kourilskjr, and A.-C. Gera pin. 1979. Cloning of the active 
thymidine kinase gene of herpes simplex virus type 1 in Esc he- 
richia coii K-12. Proc. Natl. Acad. Sci. USA 76:3755-3759. 
3. Cooe, R. D., and R. C. Mulligan. 1984. High-efficiency gene 
transfer into mammalian cells: generation of helper-free recom- 
binant retrovirus with broad mammalian host range. Proc. Natl. 
Acad. Sci. USA 81:6349-6353. 
4. Corsaro, C. M., and M. L. Pearson. 1981. Enhancing the 
efficiency of DNA-mediaied gene transfer in mammalian cells. 
Somatic CeD Genet. 7:603-616. 
5. Dick, J. E., M. C. Magtl, D. Huszar, R. A. Phillip*, tad A. 
Bernstein. 1985. Introduction of a selectable gene into primitive 
stem cells capable of long-term reconstitution of the hemo- 
poietic system of WAV’ mice. Cell 42:71-79. 
6. EgUlis, M. A., P. Kan toff, E. GUboa, and W. F. Anderson. 1985. 
Gene expression in mice after high efficiency retroviral- 
mediated gene transfer. Science 230:1395-1398. 
7. Graham, F. L., and A. J. van der Eb. 1973. A new technique for 
the assay of infectivity of human adenovirus 5 DNA. Virology 
52:456-467. 
8. Hartley, J. W., and W. P. Rowe. 1976. Naturally occurring 
murine leukemia viruses in wild mice: characterization of a new 
“amphotropic" class. J. Virol. 19:19-25. 
9. Hock, R. A., and A. D. Miller. 1986. Retrovirus mediated 
transfer and expression of drug resistance gene in human 
haemapoietic progenitor cells. Nature (London) 320:275-277. 
10. JoOy, D. J., H. Okayama, P. Berg, A. C. Esty, D. FHpula, P. 
Bohlen, G. G. Johnson, J. E. Shively, T. HunkapQUr, and T. 
Friedmann. 1983. Isolation and characterization of a full-length 
expressible eDNA for human hypoxan thine phospboribosyt- 
transferasc. Proc. Nall. Acad. Sci. USA 80:477-481. 
11. Keller, G., C Paige, E. GUboa, and E. F. Wagner. 1985. 
Expression of a foreign gene in myeloid and lymphoid cells 
derived from mullipotent haematopoietic precursors. Nature 
(London) 318:149-154. 
lla.Kwok, W, W., F. Schuenlng, R. B. Stead, and A. D. Miller. 
1986. Retroviral transfer of genes into canine hemopoietic 
progenitor cells in culture: a model for human gene therapy. 
Proc. Natl. Acad. Sd. USA 83:4552-4555. 
12. Mann, R., and D. Baltimore. 1985. Varying the position of a 
retrovirus packaging sequence results in the encapsidation of 
both unspliced and spliced RNAs. J. Virol. 54:401-407. 
13. Mann, R., R. C. Mulligan, and D. Baltimore. 1983. Construction 
of a retrovirus packaging mutant and its use to produce helper- 
free defective retrovirus. Cell 33:153-159. 
14. MlUer, A. D., D. J. JoUy, T. Friedmann, and I. M. Verma. 1983. 
A transmissible retrovirus expressing human hypo xanthine 
phosphoribosyltransferase (HPRT): gene transfer into cells ob- 
tained from humans deficient in HPRT. Proc. Nall. Acad. Sd. 
USA 80:4709-4713. 
15. Miller, A. D., M.-F. Law, and I. M. Verma. 1985. Generation of 
helper-free amphotropic retroviruses that transduce a dominant- 
acting, methotrexate-resistant dihydrofoLate reductase gene. 
Mol. CeU. Biol. 5:431-437. 
16. Miller, A. D., D. R. Trauber, and C. Buttlmore. 1986. Factors 
involved in the production of helper virus-free retrovirus vec- 
tors. Somatic Cell Mol. Genet. 12:175-183. 
17. Miller, A. D., and I. M. Verma. 1984. Two base changes restore 
infectivity to a noninfectious molecular done of Molooey 
murine leukemia virus (pMLV-1). J. Virol. 49U14-222. 
18. Panganlban, A. T., and H. M. Temin. 1983. The terminal 
nucleotides of retrovirus DNA are required for integration but 
not virus production. Nature (London) 306:155-160. 
19. Quad e, K. 1979. Transformation of mammalian cells by avian 
myelocytomatosis virus and avian erythroblastosis virus. Virol- 
ogy 98:461-465. 
20. Rasbeed, S., M. B. Gardner, and E. Chan. 1976. Amphotropic 
host range of naturally occurring wold mouse leukemia viruses. 
J. Virol. 19:13-18. 
21. Rowe, W. P., W. E. Pugh, and J. W. Hartley. 1970. Plaque assay 
techniques for murine leukemia viruses. Virology 42:1136-1139. 
22. Russell, W. C., C. Newman, and D. H. Williamson. 1975. A 
simple cytochemical technique for demonstration of DNA in 
cells infected with mycoplasmas and viruses. Nature (London) 
253:461-462. 
23. Shank, P. R„ and M. Linial. 1980. Avian oncovirus mutant 
(SE21Qlb) deficient in genomic RNA: characterization of a 
deletion in the provirus. J. Virol. 36:450-456. 
[374] 
Recombinant DNA Research, Volume 12 
