?7 
Btl*»4,dd SIX'- 
LTR N«o SVsri J'SS LTK 
FTf I Construction of the 4JT oell line. The SV(B) plasmid 
vector *u cleaved with Xhol, filled in with KJenow and treated 
with calf intestinal phosphatase (C1P). The pADA211* plasmid 
containing a full-length human ADA* cDNA was d caved with 
£coRJ and Dde I, thus renewing the 3' polv(A) addition site, filled 
in with KJenow and blunt-end ligated to the SV(B) vector. The 
"SVBADA2I1 p lasmid was isolated by banding in CaQ, and 10 tig 
purified DNA was used to transform PA-12 (ref. 9) cells by CaPO« 
precipitation. Supernatant from these cells, containing the 
transiently expressed SVBADA2II virus with an amphotropic 
envelope, was collected at 44 h and used to infect #, cells. These 
cells were placed in Dulbecoo's modified Eagle's medium 
(DMEM) with 10% PCS containing G41I (300 |sg ml* 1 ). Ninety- 
six G4|l-resistant dooes were selected and plaoed in UAAU 
(l.l nM adenosine, 30 mM alanosine, 1.0 mM uridine) plus 30 nM 
dcoxycoformycin (dCf). Two cell lines, 4.1T and 4_2T, were able 
to grow in this selective medium, and 4.2T had both higher 
expression of human ADA and higher litre virus production. LTR, 
long terminal repeat. SVori, SV40 origin of replication; pBRori, 
origin of replication from pBR322; J’SS, 3' splice site; kb, kilobaaaa 
Recombinant DNA Research, Volume 12 
[ 199 ] 
