Genetics: Kantoff cl til. 
Prov. Null. Acad. Sci. USA HJ (IV86) 6565 
1 2 3 4 5 6 7 89 10 
— 23.1 
9.4 
66 
— 2 3 
— 2.0 
Fie. 2. Southern blot of ADA-deficient and A DA -deficient. 
SAX-transduced T- and B-cell lines. Thirty micrograms of genomic 
DNA was loaded per lane except for lanes 2 and 7. which received 
10 lit Lanes l-V Sat l-digesled DNA from TJF-2 (lane II. 
GM4258A (lane 21. and GM275 (lane .1) cells transduced with SAX 
virus and selected with G41K Lane 4: SAX plasmid DNA (30 pgl 
digested with Sac I. This amount of plasmid gives a band intensity 
equivalent to “1 gene copy per genome. Lane 5: Sac l-digested DNA 
from uninfected TJF-2 cells. Lanes 6-8: CrxRI-digested DNA from 
TJF-2 (lane 6). GM4258A (lane 7). and GM2756 (lane 8) cells 
transduced with SAX virus and selected with G418. Lane 9: SAX 
plasmid DNA (30 pgl digested with £r«RI. Lane 10: £roRI-digested 
DNA from uninfected TJF-2 cells. Markers at right show migration 
positions and si/es (kh) of //mdlll fragments of phage K DNA. 
comparable to the ADA ‘ B- and T-cell normal controls (lanes 
3 and 5). Therefore, the transduced ADA-deficient T and B 
cells produce apparently normal ADA enzyme. The produc- 
tion of enzymatically active protein is not due to the activa- 
tion of the endogenous mutant ADA gene as a nonspecific 
consequence of retroviral infection, since cells transduced 
with a vector similar to SAX but not containing the ADA gene 
(viz., the parental N2 vector) do not produce enzymatically 
active ADA protein (Table 1). 
ADA enzymatic activity in lysates of the T- and B-cell lines 
was quantitated by measuring the conversion of [ u C)adeno- 
sinc to [ ,4 C|inosine (Table 1). ADA-deficient T- and B-cell 
lines produced » 19f of the adenosine-deaminating activity of 
normal T and B cells, in agreement with previous reports (27, 
1 2 3 4 5 6 7 
• t Y t 
Fig. 3. Starch gel electrophoresis of ADA-deficient and ADA- 
deficient. SAX-transduced T- and B-cell lines Lysate from 2 x 10* 
cells was added per lane. In situ chemical staining for human 
isozymes of ADA was done according to (he method of Spencer ct 
at. (25). Lane 1: ADA-positive B-cell line VSD-O. Lane 2: ADA- 
deficient B cells (GM2756) Lane 3: ADA-deficient B cells (GM27.S6) 
transduced with SAX virus and selected in G-»18 Lane 4: ADA- 
deficienl T cells (TJF-2) Lane 5: ADA-deficient T cells (TJF-2) 
transduced with SAX virus and selected in G41X, Lane 6: blank. 
Lane 7: ADA-psisitivc T-cell line CKM. 
Table 1. ADA activity and susceptibility to inhibition by 
2'-deoxyadenosine of normal ADA-positive. ADA- 
deficienl. and SAX-transduced T and B cells 
Cell type 
ADA activity* 
IC„ I .’ mM 
T-cell lines 
HM (normal ADA') 
413 2 55 
2940 
TJF-2 (ADA ) 
Not transduced 
4.7 2 1.5 
100 
SAX-transduced 
>C 
** 
41 
r** 
-r 
280 
SAX-transduced. selected 1 
1020 2 136 
2600 
SAX-transduced. selected;* 
♦ 10 m-M 2’-deoxycoformycin 
<1.0 
<100 
N2-transduced 
4.5 
110 
B-cell lines 
JM (normal ADA*) 
454 2 119 
8100 
GM2756 (ADA ) 
Not transduced 
2.7 2 0.4 
210 
SAX-transduced 
314 2 99 
1100 
SAX-transduced. selected* 
1250 2 134 
7400 
'The |‘*C)adenosine assay for ADA activity was performed essen- 
tially as described by Van der Weyden and Bailey (24). Results are 
expressed as nmol of inosine produced per min per 10* cells (mean 
2 SEM for 5 determinations, except N2-transduced TJF-2. for 
which the value is the result of a single determination). 
’Concentration of 2'-deoxyadenosine that inhibits incorporation of 
| *H (thymidine to 5091 of control value after 24 hr of culture. 
(Grown for 3 weeks in medium containing G4I8 at 1 mg/ml. 
28) in which a non-ADA aminohydrolase was detected in 
ADA-deficient cells with of normal activity. After 
introduction of a normal ADA gene by the SAX vector, these 
cells produced ADA activity at levels approximately half that 
of normal cell lines (Table 1). The expression of the intro- 
duced human ADA gene in human T and B cells is dependent 
upon the type of promoter used to facilitate transcription of 
the ADA gene. The ADA vector SAX. containing the early 
promoter of SV40 (a virus with tropism for primate cells), 
produced a considerably greater increase in ADA activity in 
TJF-2. GM2756. and GM4258A cells than similar vectors 
promoting ADA gene transcription with either the mouse 
metallothionem or the Moloney LTR gene promoter (data not 
shown). 
The T and B cells transduced with the SAX vector also 
express a second enzyme, neomycin phosphotransferase (the 
product of the neo* gene), at levels adequate for selection in 
medium containing the neomycin analogue G418. Growth of 
cells in the presence of G418 allows the evaluation of ADA 
expression in an enriched population of transduced cells 
because cells do not survive in the presence of G418 unless 
they express the neo* gene. Selection of the SAX-transduced 
cells with G418 (1 mg/ml) resulted in cell populations with 
levels of ADA activity in the normal range (Table 1). 
Protection of SAX-Transduced TJF-2 Cells from 2'- 
Deoxy adenosine Toxicity. Proliferation of ADA-deficient lym- 
phocytes is inhibited by much lower concentrations of 
2'-deoxyadenosine than is the proliferation of normal lym- 
phocytes (29). Populations of ADA-deficient T and B cells 
that were transduced with the SAX vector were tested for 
growth inhibition in the presence of 2’-deoxyadenosine. Both 
T and B cells treated with this vector showed a substantially 
increased resistance to 2’-deoxyadenosine (Fig. 4 and Table 
1). SAX-transduced cells isolated after selection with G418 (1 
mg/ml) were fully restored to normal levels of resistance to 
2'-deoxyadenosine toxicity. Addition of 2'-deoxycoformy- 
cin. a specific inhibitor of ADA activity (30). rendered these 
transduced cells sensitive to 2'-deoxyadenosine. demonstrat- 
ing that the resistance to 2 -deoxyadenosine of the SAX- 
transduced cells is. in fact . due to the production of functional 
ADA enzyme activity (Table U. 
Recombinant DNA Research, Volume 12 
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