Table 2. Successful Transfer and Expression Lsing N2 (of N2-like> Retroviral Vectors iaio 
Hemniopovetic Precursors of Various Sperm 
SPECIES 
VECTOR 
ASSAY 
REF. 
MCuSd 
N2 
CFU-S. mature celts 
(5) 
N2 
CFU-GM. mature cells 
(14) 
Neow 
GEMM. CFU-GM. BFU-E 
(4) 
Dog 
N2 
CFU-GM 
(16); a 
Human 
N2 
GEMM. CFU-GM. BFU-E 
(9)b 
N2 
GEMM. CFU-GM 
b 
N2 
CFU-GM. BFU-E 
c 
SAX 
ADA' T celts 
(13) 
Monkey 
N2, SAX 
CFU-GM. mature ceils 
a 
Sheep 
N2 
GEMM. CFU-GM. BFU-E. CFU-E 
e 
N«Ow <s a vocio r similar lo N2. as described in (4). SAX cs a VIP vector derived from 
N2 contauung iri* human adenosine deaminase cONA regulated by an SV40 pro- 
moter (13). 
a) Lot* roe. C . Egbtis. M et al. unpublished observations 
b) Dupree. J.. personal communication; Bernstein. A_. personal communication. 
Humphries RK. personal communication. 
C) Egi'tis. M et ai.. unpublished observations 
d) Kantoft. P.. OReiRy. R.. Nienhws. A. el al.. unpublished observations 
e) Zan ( ani. E. Kant oh. P. Flake. A et al.. submitted lor pubbcalon 
N2 
s* * • 
“l— — 
t 
p 
• 
p * (C 
■ — L - L tel — “1 
1 f 
ltr s * 
NEO" 
1 ^ J 
LTR 
SAX 
S- P p 
1 **"* ‘ ' 
c 
• 
p 
* * » 1C s« 
: "i 
I.TR S » 
NEO" 
■ J 
SvaO MXOA LTR 
1 • 1 
1 
a 1 a 1 a I a 1 
O 
o 
u* 
o 
I 5 
?0 
26 30 3 S 4 0 4 & 50 55 60 
SCAtE IN *0 
Figare 10. Map of SAX vector. SAX was made (13) by interims an SV40- promoted human ADA 
cDNA into the previously described (5) parental vector N2. The following regions arc indicated 
0-1. J and 4. 7-J. 5 kb. Moloney murine leukemia virus sequences. 1 .5-2. S and 4 6-4 " kb. neomycm- 
resistancc gene fNeo*) from TnJ transposori (the hatched area is the coding sequence); 2.9- 3.3 kb. 
Kpo I -Hind III fragment of the SVaOearty promoter; 3.3-4 6 kb. human ADA cDNAthADA. black 
boa); LTR. viral long terminal repeat; V. nral packaging signal Restriction sites; S. Sac 1; P. Pst 
l;E. EcoR!;C. Clal. 
monkeys and humans have been suc- 
cessfully infected, with significant ex- 
pression of the Neo* and ADA genes 
(13. 14). Bone marrow of humans, 
dogs, sheep and monkeys have been 
infected and assayed in vitro. Produc- 
tive transfer of exogenous genes has 
been achieved with each species tested, 
although, in general, at efficiencies 
lower than those found for mice. Suc- 
cessful transfer and expression of genes 
Voi 4. No 6(1986) 
into bone marrow in vivo has also been 
obtained in both sheep and non-human 
primates (14). Extensive studies are now 
underway to increase the overall effi- 
ciency of gene transfer in these and 
other large animals. 
PERSPECTIVES 
Retroviral vectors have proven to be 
an efficient means of gene transfer. 
They will undoubtedly be applied to a 
wide range of genetic studies of 
eukaryotic cdls. Even more exciting is 
the possibility of their use for the treat- 
ment of human genetic disease. 
However, many aspects of the structure 
and function of retroviruses must still 
be elucidated to optimize them as vec- 
tors. Particularly in the use of retroviral 
vectors for gene transfer into large 
animals, problems remain in the effi- 
ciency of infection of pluripotent stem 
cells and in the long-term stability of ex- 
pressed genes. I f these problems can be 
solved, then the next few decades could 
well see retroviral vectors at the heart of 
a revolution in the medical treatment of 
genetic disease. □ 
REFERENCES 
1 Anderson. W.F. 1984 Prospects for human 
gene therapy Science 226:401-409 
2. Cepko. C-L-, B E. Roberts and R.C. 
Mulligan. 1984 Construction and applica- 
tions of a highly transmissible murine 
retrovirus shuttle vector. Cell 37: 1053- 1062. 
3 Dnytou. A.I.. J.C. Sodroski. CA. Rosea. 
W -C. Cob and W .A. Haseltiae. 1986. The 
trans-activator gene of the human T cell tym- 
photropac virus type III ts required for replica- 
tion. CeD 44.941-947. 
4 Dvck. J.E.. M.C MagU. D. Huszar. R.A. 
Pkdkpi and A Bentsina. 1983. Introduction 
of a selectable gene into primitive crib capable 
of long term reconstitution of the hemopoaeuc 
system of W/W v mice Cell 42:71-79. 
5. Egiitis. M.A.. P. kanioff. E. Cdboa and 
W.F. Anderson. 1985. Gene expression in 
mice after high efficiency retrovtral-mcdiated 
gene transfer. Science 2J0~ 1 395-1398 
6 Emermaa. M. and H.M. Terrua. 1984 Genes 
with promoters in retrovirus vectors can be 
independently suppressed by an eptgenetic 
mechanism. Cell 39-459-467. 
7. Fried man. R.L 1985 Expression of human 
adenosine deaminase using a transmissible 
munne retrovirus vector system. Proc. Natl. 
Acad. Sci USA 42:703-707. 
8 Hayward. W.S.. B.G. >eri and S.M. Asxrin. 
1981. Activation of a cellular oocogene by 
promoter insertion in AJ_V -induced lymphoid 
leukosis. Nature 290:475-180. 
9 Hock. R.A. and A.D. Miller. 1986. 
Retrovirus mediated transfer and expression 
of drug resistance genes in human 
haemopoietic progenitor cells. Nature 
320:275-277. 
10 H-nng. L-S.. J. Park and E Gdboa. 1984 
Role of in tr on -contained sequences in the for- 
mation of the Moloney munne leukemia virus 
env mRNA Mol. Cdl Biol 4 2289-2297. 
11 Joyaer. A.L. and A. Berasieia. 1983. 
Retrovirus transduction: Segregation of the 
viral transforming function and the herpes 
simplex virus tk gene in infectious Fnend 
spleen focus-forming virus thymidine kinase 
vectors Mol Cell Biol 3:2191-2202 
BioTechmques 1 
Recombinant DNA Research. Volume 1 2 
[233] 
