Overview 
Table 1. Summary of Spleen Focus Analysis From Bone Marrow Infections 
-IL3 
+ IL3 
Percent 
Percent 
Hours of 
Average 
Foci 
0 DNA 
DNA 
Average 
Foci 
DNA 
Cell 
Culti- 
Initial 
Spleens 
Total 
Foci per 
Ana- 
Postlve 
Positive 
Spleens 
Total 
Foci per 
Ana- 
1 DNA 
Positive 
Line 
vation 
Titer 
Studied 
Foci 
Spleen 
lyzed 
Foci 
Studied 
Foci 
Spleen 
lyzed 
Positive 
Foci 
F-5B 
24 hr. 
1.2x10* 
2 
10 
5.0 
9 
7 
78 
8 
94 
11.8 
49 
42 
86 
48 hr. 
2.0x10* 
1 
8 
8.0 
8 
6 
75 
8 
71 
8.9 
18 
14 
78 
are easily recovered after co-cultivation. 
The hematopoietic cells are then in- 
troduced into a recipient animal by in- 
travenous injection. Space in the he- 
matopoietic system to receive the 
vector-treated marrow must be made, 
usually by lethally irradiating the 
recipient. 
Using this protocol, highly efficient 
introduction of exogenous genes into 
hematopoietic progenitor cells have 
been achieved. Experiments, such as 
those presented in Table 1 , have shown 
that greater than 85^o of spleen focus 
stem cells (so called CFU-S) can be in- 
fected with the N2 vector (5). Viability 
of the stem cells does not appear to be 
impaired, and the vector has been 
shown to integrate in its intact form. In 
some cases, relatively low efficiencies 
have been improved by pre-selection of 
infected marrow prior to introduction 
into a recipient mouse (4, 15). Advan- 
tage is taken of the presence of a selec- 
table gene in the vector to enrich the 
total population of marrow for cells 
containing the vector. Incubation in 
high concentrations of the selective 
agent for one or two days between in- 
fection and transplantation has im- 
proved the recovery of infected cells be- 
tween three- and ten-fold (4, 15). 
Not surprisingly, the titer of the vec- 
tor is of great importance in the suc- 
cessful transfer of exogenous genes in- 
to hematopoietic progenitor cells. In 
general, titers of at least 10 4 cfu/ml 
seem to be required (Fig. 8). Although 
vector instability appeared to be a prob- 
lem in early efforts at gene transfer in- 
to bone marrow (12), recent experi- 
ments with helper-free vectors show this 
to be a reduced concern (4, 5, 15). 
Minimizing the viral sequences retained 
in the vector reduces the likelihood of 
recombinatorial disruption of the 
vector. 
Expression of transferred genes in 
tissue culture cells is very efficient. At 
present, N2 is the only vector that has 
been consistently shown to exhibit effi- 
cient expression (of the Neo R gene) in 
the mature hematopoietic cells of long- 
term reconstituted mice (5, 15). Drug 
resistant colony forming progenitor 
cells and enzymatically active protein 
can be obtained from mice several 
months after gene transfer (Fig. 9 -right 
panel). Thus, these initial mouse studies 
have borne out the potential of 
retroviral vectors for the high efficiency 
transfer of intact, functioning vectors 
stably into bone marrow cells. 
We have added an SV40-promoted 
human adenosine deaminase (ADA) 
gene to the N2 vector as shown in Fig. 
10. This VIP vector, called SAX (for 
SV40, human ADA, inserted into the 
Xhol site), has been used to introduce 
the ADA gene into the hematopoietic 
cells of several species other than mouse 
(Table 2). Lymphocyte lines from both 
Figure 8. Effect of titer on Infectivlty of murine 
CFU-S. Bone marrow was treated with vectors 
of the titers shown. Titer is expressed by the 
number of drug resistant colony forming units 
(efu) per ml assayed in vitro on a tissue culture 
cell line (usually 3T3 cells, a murine fibroblast 
cell line). A titer of 10* cfu/ml seems critical for 
successful infection. Interleukin- 3 (IL-3), whjch 
is a growth stimulating factor, may improve in- 
fection slightly by stimulation of the bone 
marrow. 
neo r phosphotransferase gene expression 
IN N2 - TREATED IRRADIATED MICE 
in to Oav Soieen Foci ,n 4 Moo,h ncconci.t w i« M.ce 
Figure 9. Neo* phosphotransferase gene expression in N2-tre*ted irradiated mice. Bone marrow 
cells were infected with the N2 vector as shown in Fig. 7. In the left panel, individual spleen foci 
were isolated and assayed for the presence of the Neo*-gene-coded phosphotransferase (see Ref. 
5). In (his case, all six individual foci had detectable levels of enzyme activity, although amounts 
varied. * . positive tissue culture control; -, uninfected spleen foci cells; Pool, activity of 8 pooled 
foci from one spleen. In the right panel, four mice were analyzed four months after receiving suffi- 
cient numbers of treated bone marrow cells to reconstitute their entire hematopoietic system (see 
Fig. 7). In three of four mice (#1 , 3, and 4), low levels of enzyme activity were still detectable in 
the bone marrow (M). In one of these three mice (#3). activity was also detected in the blood (B). 
510 BioTechniques 
Vol. 4. No. 6 (I986> 
[232] 
Recombinant DNA Research, Volume 12 
