abundance of proviral sequences by Southern blot analyses (see Fig.3 for example). This has 
been repeated two times with essentially identical results. The copy number of intact provirus 
was 1 copy/cell on both occasions and rearranged proviral sequences were never detected. 
An alternative and complementary approach is to assess efficiency of gene transfer based 
on transgene expression. A viral stock is exposed to a subconfluent plate of diploid fibroblasts 
from a patient with FH for 12-16 hrs in the presence of polybrene (8 ug/ml). When 
confluent, the cells are incubated with fluorescent labeled LDL for 4 hrs and visualized by 
fluorescence microscopy. The relative number of fluorescent cells provides a minimal estimate 
of gene transfer. This analysis has been performed four different times with virtually identical 
results. The relative number of fluorescent cells varied from 50-80% in these experiments. 
The #132-10 viral producer was analyzed on three separate occasions for the production of 
replication-competent virus of ecotropic or amphotropic host range using the previously 
described lacZ mobilization assay. A brief description of the method is provided below. The 
viral supernatant is exposed to a subconfluent plate of the cell line, 3T3BAG, for 12-16 hrs in 
the presence of polybrene (8 ug/ml). The 3T3BAG cell line is a clone of NIH3T3 cells which 
harbors a single copy of a lacZ containing provirus. The infected 3T3BAG population is 
propagated in culture through two passages over a ten day period. A supernatant is then 
harvested from the 3T3BAG population and exposed to a subconfluent plate of NIH3T3 cells for 
12-16 hrs in the presence of polybrene (8 ug/ml). When confluent, this infected population of 
cells is analyzed for the presence of the lacZ provirus using the chromogenic cytochemical 
assay. The packaging cell line ¥2 which packages the 'F- genome at a low level is always used as 
a positive control. Supernatant from #132-10 harvested three different times following 
initial seeding (up to one month) was analyzed using the lacZ mobilization assay (Experiment 
#1- duplicates, Experiment #2- duplicates, and Experiment #3- quadruplicates). Each 
experiment scored negative. 
5. Production of the LDL receptor viral supernatant 
Day 0 
1 . Make complete media by filtering the following components through a 0.45|im one 
liter filter: 1000 ml of high glucose DMEM and 100 ml of bovine calf serum 
2. Use a 10 ml pipette to remove 10 ml of media from each liter to test for sterility. 
3. All media will be stored at 4 °C. 
4. To thaw the seed lot of #132-10, take five cryovials of each from the -135 °C and put 
a 37 °C water bath. 
5. Use a 10 ml pipette and add 10 ml of complete medium to 5 x 10 cm tissue culture 
plates. 
6. Use a 1 ml pipette to transfer the cells from each cryovial to a plate. 
7. Place the plate at 37 °C, 5% CO2. 
8. Two hours later, aspirate off media with a pasteur pipette and add fresh complete 
medium with a 10 ml pipette and return plate to 37 °C, 5% CO2. 
Recombinant DNA Research, Volume 15 
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