Biochemistry: Yu tt al. 
Pruc. Nail. Acad. Sci. USA S3 (I9S6) 3197 
A 
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B 
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Fic. 4. Expression of the MT-c-/»i hybnd gene in NIH JT) cells 
introduced by infection with a SIN vector. (A) The MT-c-fos DNA 
construct (27) consists of the human MT promoter linked to the 
promoterless mouse c-/<*i gene, which contains three tntrons and a 
polyadcnylylalion signal l24). A 4,V-kb £c oRI/V/mdlll DNA frag- 
ment was cloned into the 0</»iHI site of TK-N. a SIN vector 
containing the neu'-gene expressed from the herpesvirus TK promoter. 
The MT-c-/m construct was inserted in TK-N in the antisense onen- 
tation This construct. F-TK-N. generates three transenpts initiated 
from the TK. MT. and viroJ promoters. (B I Analysis of provjraJ DNA 
in cells infected with virus derived from F-TK-N. Virus derived from 
independently transfected *2 cells was used to infect NIH 3T3 cells, and 
analysis of the cellular DNA by Southern blotting was performed as 
described in Material t and Mnlutdt and in the legend to Fig 2. Cellular 
DNA was digested with Nhe I. which has a recognition site within the 
viral LTRs (see Fig. I) as well as within the c-/r>i gene generating a 
3.5-kb and a 4.5-kb DNA fragment as sho»vn in A . If the deletion in the 
J' LTR of F-TK-N is transferred to the 5’ LTR of the provirus, a 3.2-kb 
fragment (300 bp shorter) will be present in the infected cell. The DNA 
bkx was hybridized with a ' : P-labeled c-/oi-contaimng plasmid. Lanes 
I and 3. two cell lines infected with virus preparations derived from two 
independently derived transfected cell lines; lane NIH 3T3. uninfected 
cells: lane P. F-TK-N plasmid. The 4.5- and 3.5-kb DNA fragments in 
F-TK-N as well as the 3.2-kb DNA fragment in the infected cells is 
indicated by the arrows. The third band in lane P migrating below the 
3.5-kb band represents plasmid sequences. The two upper bands 
present in all lanes, including the uninfected NIH 3T3 cells, are 
endogenous c-fos sequences. The origin of the additional band in lane 
3. which is 6-7 kb long, is not known and can also be detected as a faint 
band in uninfected NIH 3T3 cells. (C) Analysis of RNA in cells infected 
with viruses derived from F-TK-N was performed as described in Fig. 
3. Lanes 1. 2. and 3. three cell lines derived by infection with viruses 
onpnating from an independently transfected <*2 cell. K„ is a cell line 
denved by transfection with the MT-c-/«j hybnd gene alone (25). 
Hybndization was performed with a <: P-labeled c-/os-specific probe 
and a ' : P-labeled ras-specific probe, which serves as an internal control 
for the amount of RNA loaded in each lane. * and - indicate cells that 
were or were not incubated for 6 hr in the presence of 5 *iM CdCI- before 
isolation of RNA. 
virus (called F-TK-N) from three independently derived 
transfected colonies was used to infect NIH 3T3 cells. The 
titer of the three virus preparations was » 10' colony-forming 
units/ml. 1/ 10th the liter of the parent vector TK-N. The 
structure of the proviral DNA in two cell lines generated with 
two different virus preparations was analyzed by Southern 
restriction analysis as shown in Fig. 4fl. This analysis has 
shown that the provirus in the infected cells is intact and that 
the deletion from the 3’ LTR was transferred to the 5' LTR. 
as stated above. Analysis of cytoplasmic RNA in the infected 
cells by RNA blotting and hybridization with a c-/o.t-specific 
probe, as shown in Fig. 4C. reveals the presence of only one 
RNA transcript. This RNA species comigrates with the 
2300-nucleotide-long authentic c-fos mRNA present in cells 
transfected with the hMT-c-fos DNA construct alone (Fig. 
4 C. ACJ. No readlhrough transcripts. «7500 nucleotides long 
extending from LTR to LTR (see Fig. 4A). can be seen. 
Expression of the hMT-c-/w gene is induced by cadmium in 
cells transfected with the hybrid DNA construct (ref. 25: Fig. 
4C. K„). Expression of the same hMT-c-/w transcript in cells 
infected with virus derived from F-TK-N is also inducible by 
cadmium (Fig. 4C). In cell line 3. the extent of cadmium 
induction is similar to (he cell line transfected with the 
h.MT-c-/f>i gene alone (Fig. 4 C. K a ) and in cell lines 1 and 2. 
the extent of induction is lower. 
DISCUSSION 
In this report, we have described a retroviral gene transfer 
system designed for the efficient transduction of whole genes 
based on so-called SIN vectors enabling the expression of the 
transduced gene without the interference of a readlhrough 
transcript originating in the viral LTR. The main feature of 
SIN vectors is a deletion introduced in the 3' LTR. which is 
transferred to the 5" LTR in the proviruses integrated in the 
target cell, eliminating the virus's transcriptional activity. 
The development of useful retrovirus-derived vectors for 
the transduction of whole genes has been plagued by several 
problems. In many cases, it was shown that the infected cells 
selected for the expression of the selectable gene contained 
rearranged proviruses from which large portions of the 
transduced gene had been deleted (26-28). It is possible that 
the instability of these vectors stems from the fact that a 
whole gene is inserted within the retroviral genome resulting 
in unfavorable interactions between the two transcription 
units. Another disturbing aspect of retroviral-based gene 
transfer systems is the presence of two functional LTRs, 
resulting in an RNA transcript extending through the 
transduced gene. This, as well as the presence of enhancer 
sequences within the LTRs. is likely to influence the overall 
levels and the regulated expression of the transduced gene. 
In the SIN vectors described in this study, the deletion of 
299 bp removes most of the two 72-bp repeats that constitute 
the viral enhancer and the promoter region but leaves the 
TATA box intact (20. 21). In the design of the SIN vectors, 
we were careful not to remove all U3 sequences up to the 3’ 
end of the R region because it was suggested (29. 30) that this 
region may be involved in the polyadenylylation process. On 
the other hand, removal of only the enhancer region — for 
example, by a deletion of a Pva ll/Xba I fragment (Fig. 1) — 
may not suffice because it is reasonable to expect that in 
many cases the internal promoters carrying their own en- 
hancers will activate the enhancerless LTR. In fact, it is 
possible that the low level of LTR-driven transcription seen 
in Fig. 3 (0.1-19J) is due to such an effect mediated by the 
SV40 enhancer, while the apparent absence of such LTR 
transcription in the MT-coniaining proviruses may be due to 
the lower efficiency of the enhancer activity associated with 
this promoter. It is also possible that the low level of 
transcription originating from the viral LTR seen in Fig. 3 
occurs due to recombination in the process of infection. 
Recombinant DNA Research, Volume 12 
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