4.4.6 Precautions: 
Precautions against recombination and formation of a hybrid human bovine virus: 
Homology comparison of BPV1 with human papilloma virus (HPV) sequences in the genbank data base 
indicates HPV 6 and 31 as the human subtypes with the highest homology to BPV1 (Figure 9; see 
Appendix D). Inspection of the aligned sequences shows, despite similar overall organization, only 
relatively limited homology. The longest stretch of uninterrupted sequence identity corresponds to 14 
nucleotides, probably insufficient to support homologous recombination. Papilloma viruses are highly 
species specific (4). Therefore even in the event of a homologous recombination and generation of a 
hybrid virus it is highly questionable whether such a virus would be infectious for human cells. 
Nonetheless we are taking the following precautions. Human tumor cells to be transfected will be 
first screened by Southern dot blots for the presence of human papilloma virus sequences. This 
procedure will cover HPV type 6, 11, 16, 18, 31, 33, 35 which encompass the majority of all 
occurrences. Positive cells will not be used for transfection. 
In our studies pBMG-Neo will be used to transfect tumor cells for the expression of lymphokines. 
Tumor cells are fully transformed cells. Transfection of murine and human tumor cells does not 
decrease the doubling time (Table 1, see Appendix C) and transfected cells do not appear to be more 
transformed than untransfected cells in phenotype and morphology. As precaution against tumor growth 
at the injection site, we are planning to irradiate tumor cells in such a way that their replication is 
impaired, but that they continue to produce the transferred gene products (lymphokines). The occasional 
surviving cell even after irradiation is expected to be eliminated by the ensuing immune response at the 
injection site due to lymphokine production. 
The potential benefit of an induced immune response in our opinion by far outweighs the small risk 
of surviving tumor cells. 
4.5 Transfection of SCLC 
Plasmid BMG-Neo is a member of the newest generation of expression vectors (44) that affords 
selection of transfected cells by G418 through its content of a Neomycin resistance cassette transcribed 
under the influence of the thymidine kinase promotor. The cDNA of interest is expressed under the 
ubiquitously active metallothionein promotor. Most important for high level and stable expression is 
the multicopy number of the plasmid (20-100 per cell) which is maintained as an episome in the cell 
(45). The latter property derives from the 5.5kb viral (69%) fragment derived from the bovine 
papilloma virus type 1. Finally the plasmid has the pBR322 origin of replication and ampicillin 
resistance for maintenance in E. coli. 
A bottleneck in the expression of eukaryotic genes often is the difficulty to establish the DNA to 
be expressed in the appropriate cell. One way to achieve this was through the development of retroviral 
vectors. By generation of infectious but unproductive viruses with the aid of a helper virus containing 
(packaging) line, DNA is transferred to cells by the broad specificity of the virus particle. Elegant 
though this technique is, it is limited by the size of the inserted DNA, by the inability to control the 
locus of insertion into the genome with its potentially negative consequences such as low level 
expression, and by the small but worrying risk of generating complete infectious retroviruses during the 
packaging process. 
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