Galpin/DA/N2 IIIBenv 
2.1.1 Sequence Analysis of the Vector 
A scale diagram and functional description of the components of the N2 IIIBenv 
provector plasmid are included in Appendix A. 
Also provided in Appendix A is the complete report from Lark Technologies 
including a nucleotide sequence analysis, a restriction enzyme analysis and the 
amino-acid translation of the N2 IIIBenv provector. One nucleotide difference was 
observed between the HIV-1 IIIBenv sequences from that expected. This change does 
not change the amino acid sequence. 
2.2 PACKAGING CELL LINE 
A novel amphotropic packaging cell line (designated DA) was created from a 
parental canine cell line (D-17, ATCC #CCL183) by sequentially transfecting the genes 
for the carrier structural proteins, gag/pol and env <am (amphotropic envelope) into the 
parent cell line. The structural gene, gag/pol, was derived from the Moloney murine 
leukemia retrovirus 27 (MoMLV) whereas the envelope gene, env' 1 ™, was derived from the 
murine leukemia retrovirus MLV 4070A 28 . The packaging cell line continuously 
expresses the corresponding MLV structural proteins and as a result, produces "empty" 
virion particles 29 . When a retroviral provector, such as N2 IIIBenv, is introduced into the 
packaging cell line, the MLV structural proteins package the retroviral vector genome 
into the virion particle and nascent retroviral vector accumulates in the cell medium. 
2.2.1 Parent Cell Line 
The canine cell line D-17 was originally chosen as the parent for the packaging 
cell line because its genome was expected to lack homology to murine C-type 
retroviruses. This was confirmed by Southern blot analysis which showed no specific 
cross hybridization between D-17 genomic DNA and the MLV gag/pol and env am 
used in the construction of the packaging cell line. The parent cell line has also been 
27 Miiler, et al., Mol. Cell. Biol. 5:431-443, (1985). 
28 Chattopadhyay, et al., J. Virol., 39 *777-79 1 , (1981). 
29 Cometta, et al., Human Gene Therapy, 2:5-14, (1991). 
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