retroviral gene transfer and expression of a foreign gene in a mixed population 
of differentiating but non-transformed hematopoietic cells. It was not 
possible to determine, however, whether any of the Neo R -transduced colonies had 
originated from an uncommitted hematopoietic stem cell. 
Double expression vectors carrying two genes are capable of transmitting 
these genes Intact and producing functional proteins from full-length or 
appropriately spliced mRNA transcripts. One such vector. pZIP-ADA (Valerio et 
al.. 1984) was used to introduce a human ADA cDNA and Neo R gene to NIH 3T3 
cells. After several cell passages in the absence of G418 selective medium, 
cells still produced human ADA and were G418-resi stant indicating efficient and 
stable expression of the provlral DNA sequences in this transformed cell line. 
A similar double expression vector containing a cDNA for human 
□ 
phenylalanine hydroxylase (PAH) and the Neo gene both under transcriptional 
control of the viral LTR sequence contained In the vector was developed by 
Ledley et al.. (1986). Variable expression of human PAH was detected in both 
NIH 3T3 cells and a mouse hepatoma cell line after infection with the human 
D 
PAH/Neo vector and selection In G418-containing medium. 
In another series of double expression vectors a human ADA cDNA was 
Inserted with or without an internal SV40 promoter along with a mutant 
dihydrofol ate reductase (DHFR*) gene as a dominant selectable marker (Williams 
et al., 1986). Following infection of a mouse pre-B cell line (70Z) with this 
vector, human ADA activity was greater than endogenous murine ADA activity 
after selection with methotrexate to eliminate uninfected cells not 
concomitantly expressing the DHFR gene and human ADA gene. The SV40-promoted 
ADA gene in this vector was also shown to be functionally active in an 
ADA-deficient human B -cel 1 line even in an unselected population of cells. The 
same vector was used to transmit human ADA into murine bone marrow cells prior 
Recombinant DNA Research, Volume 12 
[37] 
