and env functions on separate constructs into NIH3T3 cells. The original paper describing the 
isolation of the cell line is provided in Appendix I. The 3' LTRs of the constructs were replaced 
with heterologous polyadenylation sequences. These modifications were performed to miiiimize 
the chance that recombination could result in the production of replication competent virus. 
The plasmids used to make the packaging cell line are described in the original paper. 
The vector was cotransfected into 'F-CRIP with pSV2Neo, and stably transfected clones 
were selected in G418 (1 mg/ml). Individual clones (25 from each transfection) were isolated 
and analyzed for production of virus. The producer is maintained in Dulbecco's modified medium 
supplemented with 10% bovine calf serum. Supernatants from confluent plates of clones were 
harvested and exposed to subconfluent plates of a human fibroblast line deficient in LDL 
receptor in the presence of polybrene (8 jig/ml). Expression of wild-type LDL receptor was 
assayed in situ by incubating the cultures with fluorescent labeled LDL (14). Fluorescent 
microscopy revealed substantial activity in a subpopulation of fibroblasts from each infected 
culture. 
Freshly isolated viral supernatants were analyzed for replication competent virus using 
the previously described LacZ mobilization assay (90). NIH 3T3 cells harboring a single copy 
of a recombinant retroviral genome encoding E. coli p-galactosidase were exposed to the viral 
supernatant and maintained in culture for 7-10 days. A supernatant was harvested and used to 
infect NIH 3T3 cells which were subsequently analyzed for LacZ expressing cells using X-gal 
chromogenic assay. None of the virus producers have scored positive for replication competent 
virus or packaging of the psi genome using this sensitive assay. 
The structure of the retroviral vector used to make this virus producer is illustrated in 
Figure 2. The enhancer from CMV has been cloned in reverse orientation immediately upstream 
to the p-actin promoter. In general, the CMV containing vectors produced much higher titers of 
virus than the vectors which did not contain additional enhancers or those that contained the 
alpha-fetoprotein enhancer. 
The relative titers of several candidate producers were characterized by exposing a 
subconfluent plate of NIH3T3 cells to freshly isolated viral supernatants and analyzing the DNA 
from the infected, unselected population of cells for the presence and abundance of proviral 
sequences. A representative Southern analysis from this experiment is presented in Figure 3. 
Several infected populations demonstrated unrearranged proviral sequences at frequencies 
greater than or equal to 1 copy of provirus per cell. The vector does not contain a selectable 
marker so it is impossible to perform a limiting dilution assay. Previous experience in our 
laboratory suggests that a viral stock which transfers 2 proviral copies per NIH3T3 cell would 
be equivalent to approximately 1 to 5 x 10® cfu/ml in a limiting dilution assay. RNA blot 
analysis of total cellular RNA from the infected unselected NIH3T3 cells is presented in Figure 
4. In each case, the transcript initiated from the p-actin promoter is more abundant than the 
LTR initiated transcript. 
Viral stocks were also used to infect a diploid fibroblast cell line derived from a patient 
with FH. The infected unselected population of cells was analyzed for production of functional 
LDL receptor protein using the previously described 125 |_|_dl binding and degradation. Many 
of the transduced populations of FH cells expressed levels of LDL receptor that exceeded those 
measured in fibroblast cells derived from a normal individual as seen in Figure 5. Finally, as 
described in the initial proposal none of the viral producers passage the 4 / ' genome or produce 
replication competent virus. 
Based on the Southern and Northern experiments, as well as the 12 5|-LDL functional 
assay we have selected producer #132-10 as the one to be used in the human experiments. 
Recombinant DNA Research, Volume 15 
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