We have extensive experience with 'PCRIP as a packaging cell line in combination with 
the type of vector used to make CMV-BA-(h)LDLR (i.e., internal promoter, SD+, gag+, and 
with enhancer deletions in the 3’ LTR- see Fig. 2 for basic structure). Our experience indicates 
that this combination is effective in producing high titer virus as well as safe in terms of 
production of replication-competent virus or packaging of the genome. Between July 
14,1987 and September 7,1991, we have performed 159 transfections of retroviral vectors 
into the 'FCRIP (94 transfections) and 'FCRE (65 transfections) packaging cell lines. 
Retroviral vectors of the type used to construct CMV-BA-(h)LDLR have been used in 110 of 
these experiments. Approximately 25 stable viral producers from each transfection have been 
isolated and characterized with respect to viral titer resulting in a cumulated experience of 
approximately 3975 individual stable producers. The highest titer stable producer from each 
transfection are usually screened for replication-competent virus using the lacZ mobilization 
assay. We have never detected replication-competent virus form these cell lines. 
4. Characterization of the LDL receptor virus producing cell line 
# 132-10 
A master cell bank (MCB) of the #132-10 cell line, comprising of 100 ampules, has 
been established. We are in the process of characterizing this cell line using criteria suggested 
by the FDA in published documents. The following analyses of the MCB are underway. We expect 
these studies to be completed by November 1,1991. A brief protocol for each assay can be found 
in Appendix K. 
lest 
Performed Bv 
Sterility 
OL 1 
Cell Culture ID 
OL 
Mycoplasma 
OL 
Induction RT 
OL 
Extended XC Plaque 
OL 
Extended S+L- Focus 
OL 
MAP 
OL 
In Vivo Virus 
OL 
In Vitro Virus 
OL 
Co-cultivation with Mink Lung 
OL 
Co-cultivation with RD 
OL 
Bovine Viruses 
OL 
Porcine Parvovirus 
OL 
LDL Receptor Transduction 
H.A.L. 2 
1 Outside Laboratory 
2 Human Applications Laboratory, University of Michigan 
We have extensively analyzed the cell line used to lay down the MCB (#132-10) with 
respect to 1) LDL receptor transduction and 2) production of replication-competent 
retroviruses with ecotropic or amphotropic host range. The producer has been analyzed for LDL 
receptor transduction and helper virus on multiple occasions after it had been maintained in 
culture for various times up to 4 weeks 
Viral titer is assessed one of two ways. One approach is to harvest virus and infect a 
subconfluent plate of NIH3T3 cells for 12-16 hrs in the presence of polybrene (8 ug/ml). 
When the cells are confluent, total cellular DNA is harvested and analyzed for integrity and 
[180] 
Recombinant DNA Research, Volume 15 
