animal was demonstrated by the presence of G418-resi stant CFU-C from 
reconstituted mice and NPT activity from tissue extracts of bone marrow, thymus 
and spleen. Both types of assays require that the gene product from the 
Introduced Neo R gene be biologically active. A comparison of the normal 
activity of a bacterial gene in mammalian cells cannot be made. The presence 
of the Neo R gene product (i.e.. NPT) was not assessed in tissues other than 
those that might have been derived from bone marrow progenitor cells. 
Gene expression from the SAX retroviral vector has been studied in our 
laboratory using human ADA-deficient T and B cells (Kantoff et al . , 1986) as 
well as murine NIH 3T3 cells and monkey CV-1 cells (McLachlin et al.. 1987) in 
vitro (copies of both papers provided in Appendix B). In these studies, cells 
D 
were selected in G418-containing medium which requires expression of the Neo 
gene and from this same population of cells expression of the introduced human 
ADA gene was detected. All of the methods of assay required biologically 
active protein. Starch gel electrophoresis of whole cell extracts of 
SAX-transduced human ADA(-) cells and mouse and monkey cells demonstrated high 
levels of expression of the human ADA gene. There was also evidence for the 
appropriate post-translational modification of the primary ADA gene product 
since in the human as well as in the mouse and monkey electrophoresis 
demonstrated the three characteristic human ADA isozymes. The levels of 
expression of the human ADA gene were at least equal to the endogenous levels 
of ADA in the mouse and monkey cells after column separation of 
species-specific ADA enzymes (McLachlin et al.. 1987). In addition, the levels 
of ADA expression in SAX-transduced human ADA-deficient cells approached normal 
levels in unselected populations of cells and increased to 2-4 times the levels 
seen in normal control human cells after G418 selection for an enriched 
Recombinant DNA Research, Volume 12 
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