3196 Biochemistry: Yu el <il. 
DN A transfection procedure (15). 02 cells are a derivative of 
N1H 3T3 cells that contain a packaging deficient Mo-MuLV 
helper provirus (14). The RNA transcribed from the trans- 
fected DNA constructs is packaged into retrovirus virions 
and is secreted into the medium. The hybrid virus generated 
from the vectors shown in Fig. 2 was used to infect NIH 3T3 
cells, and cells expressing the neo' gene were selected in 
medium containing G418 (1 mg/ml) (16. 17). The titer of 
nco'-containing viruses was determined by infection of NIH 
3T3 cells with serial dilutions of the medium obtained from 
independently isolated transfected colonies. For each vector 
construct, the titer from six to eight transfected colonies was 
determined. In all cases, the highest titers of the hybrid 
viruses were similar, ranging from 2 x 10 4 to 1.0 x 10* 
colony-forming units/ml. In all subsequent experiments de- 
scribed in this paper, the virus preparation used to infect the 
cells originated from an independently transfected colony. 
Biochemical Analysis of Cells Infected with SIN Vectors. The 
structure and transcriptional activity of SIN vectors in the 
infected cells was characterized. NIH 3T3 cells (2 x 10 5 ) were 
infected with undiluted virus at 0.01-0.1 colony-forming units 
per cell and were grown for 2-3 weeks under selective 
conditions in the presence of G418. Under these conditions, 
the majority of the infected cells will contain one copy of the 
provirus (data not shown). The structure of the provirus 
integrated in the chromosome of the infected cells was 
determined by Southern blot restriction analysis (18). The 
purpose of this analysis was to determine whether the 
deletion present in the 3' LTR of the SIN vectors constructed 
in E. coli was. as predicted, transferred also to the 5' LTR of 
the provirus in the infected cell. The data are shown in Fig. 
2. Three virus preparations containing the MT promoter 
linked to the neo' gene (MT-N) and three virus preparations 
containing the S V40 promoter linked to the neo' gene (SV-N) 
were analyzed in this experiment. Each lane represents the 
DNA content of NIH 3T3 cells infected with virus secreted 
by cells originating from an independently derived trans- 
fected colony. The restriction enzyme Nlie I was used 
because it has a unique recognition site in the viral LTR 
between the 5' end of U3 and the 5' end of the deletion, 
generating a characteristic restriction fragment 3400 and 3200 
bp long when MT-N and SV-N are digested with Nlte I. 
respectively (Fig. 2). If the 299-bp deletion present in the 3’ 
LTR of MT-N or SV-N is transferred to the 5’ LTR of the 
integrated provirus, the Nlte I fragment in the infected cells 
will be 299 bp shorter. This is indeed the case in all three virus 
preparations derived from SV-N and in one of three virus 
preparations obtained from MT-N (Fig. 2). as well as from 
two virus preparations obtained from F-TK-N (see Fig. AB). 
Digestion with Tilt III-I was used to confirm that the 3’ LTR 
ofihe integrated proviruses does not differ from the original 
vectors and still contains the original deletion (data not 
shown). 
The DNA structure of proviruses originating from the two 
virus preparations M-2 and M-3 (Fig. 2) differs from the 
predicted structure. In addition to the expected band, a 
second slower-migrating band is seen, similar in size to the 
original MT-N DNA construct. The virus preparations de- 
rived from the transfected 02 cells and used to infect NIH 3T3 
cells generating the cell lines SI. S2. S3, and Ml shown in Fig. 
2 always gave rise to the same proviral structure upon 
subsequent infection of NIH 3T3 cells. This suggests that the 
rearranged viruses (M-2. M-3) arose through recombination 
during transfection, and. as indicated in Fig. 3. regenerated 
a functional 5’ LTR. Therefore, in using SIN vectors, several 
independently transfected colonies should be isolated and 
expanded to cell lines, and the structure of proviruses can be 
determined by DNA blotting as shown in Fig. 2. Virus- 
producing cell lines that generate unrearranged virus (e.g.. 
virus preparations used to infect NIH 3T3 cells in Fig. 2: SI. 
Proc. Nall. Acad. Sci. USA S3 <1986) 
cm r> cm r> 
vn I I I 
* - »s 
r * 
Fig. 3. RNA analysis in cells infected with SV-N and MT-N 
vectors. Poly(A)-containing cytoplasmic RNA from the cell lines 
described in Fig. 2 was analyzed by RNA blotting. Vector-specific 
RNA was detected by hybridization with a neo'-specific probe. The 
diagram in Fig. 2 shows the predicted sizes of the RNA transcripts 
(including the poly(A) tail] originating from the viral LTR and the 
internal promoter. The migration of mouse 18S and 28S rRNA is also 
shown (1950 and 4500 nucleotides, respectively). (Upper) An 18-hr 
exposure. (Lower) A 6-day exposure of the same gel. 
S2. S3, and Ml) are then selected for further use. providing 
an unlimited source of nonrearranging virus. 
The transcriptional activity of the proviruses was analyzed 
next. As shown in Fig. 2. the vectors MT-N and SV-N 
contain two transcriptional units. One transcriptional unit is 
initiated in the 5' LTR and the second unit is initiated in either 
the MT or the SV40 DNA fragment. Both transcriptional 
units use a common polyadenylylation signal present in the 3' 
LTR. When the deletion in the 3' LTR is transferred to the 
5' LTR in the integrated proviruses, the LTR-driven tran- 
scriptional unit should be eliminated and. if so. the infected 
cells will contain only one transcript initiated from the 
internal promoter. As shown in Fig. 3, this is indeed the case. 
Cell lines SI. S2. S3, and Ml express predominantly one 
RNA species in the cytoplasm corresponding in size to the 
RNA transcript expressed from the internal MT or SV40 
promoter. Only when the autoradiogram is overexposed is a 
faint band noticeable corresponding in size to the transcript 
initiated in the 5' LTR. The level of this transcript is 0.1-1% 
of the transcript expressed from the internal promoter. On 
the other hand, in cells infected with M2 or M3, in which an 
apparently functional S' LTR was regenerated (Fig. 2). or in 
cells infected with similar hybrid vectors containing intact 5’ 
and 3’ LTRs (data not shown), the level of transcripts 
initiated in the viral LTR is equal to or higher than that of 
transcripts expressed from the internal promoter. 
Transfer and Expression of the Inducible Hybrid Gene by 
Using a SIN Vector. The use of SIN vectors to transduce 
whole genes was demonstrated by using the hMT-c-/os 
hybrid gene in which the promoterless c -fos gene containing 
its three introns and a polyadenylylation signal (24) is fused 
to the inducible human MT promoter (25). In this experiment, 
we show that the MT-c-/or gene [4.9 kilobases (kb) long] is 
introduced into NIH 3T3 cells through a retroviral SIN vector 
without undergoing gross rearrangements, and an authentic 
c-fos RNA transcript is formed that is inducible by the heavy 
metal cadmium. Moreover, no readthrough RNA transcript 
originating from the viral LTR is present in the cells. 
As shown in Fig. AA. the hMT-c-/rw hybrid gene was 
cloned in the antisense orientation, into the Bam Hi site, in 
from of the thymidine kinase (TK)-promoted neo' gene. The 
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Recombinant DNA Research, Volume 12 
