Several subsequent studies demonstrating expression of a foreign gene in 
the whole animal have used retroviral vectors containing the bacterial Neo R 
gene (Dick et al.. 1985; Keller et al.. 1985; Eglitis et al.. 1985). In one 
series of Neo R vectors (Dick et al.. 1985). MLV-NEOI contained the SV40 early 
promoter and the NEO vector carried the mouse immunoglobulin heavy chain 
enhancer Inserted immediately upstream from the Neo R gene in the parent. 
pZ IP-NeoSV (X) . vector. These vectors were first tested by pretreating the 
donor marrow in vivo with 5-fl urouracil (5-FU) and preselecting the infected 
cells 1_n vitro in liquid culture with G418 for 48 hours prior to plating the 
cells In methyl cellulose to assay colony formation in vitro . With the NEOyv^ 
D 
vector 60-100* of the CFU-C expressed the Neo gene and were resistant to G418 
after the period of preselection. A similar protocol was followed for the 
Infection and 48-hour preselection of bone marrow cells prior to Infusion into 
lethally Irradiated mice. Preselection of Infected cells in G418 dramatically 
p 
Increased the number of spleen colonies (CFU-S) containing the Neo gene 
p 
sequences In transplanted animals. Expression of the Neo gene was examined by 
colony formation J_n vitro in the presence of G418 of bone marrow stem cells 
from reconstituted mice taken at 11. 13. and 17 weeks post-transplant. In 
contrast to the level of expression seen in colony forming cells infected with 
the NEO^vector and cultured l£ vitro directly, culture of bone marrow 
progenitor cells from reconstituted mice showed that only a portion of the 
CFU-GM (2 to 20*) were resistant to G418 when assayed at various times after 
the Initial Infection and transplantation. This observation as well as others 
has lead to the conclusion that differentiating cells In vivo provide a less 
permissive environment for the expression of Introduced genes. 
p 
The Neo gene has also been inserted into mouse bone marrow cells using 
the Mo-MuLV based vectors N2 and N3 (Keller et al.. 1985; Eglitis et al., 
1985). The N3 vector is similar to N2 except for a smaller fragment of intact 
[ 43 ] 
Recombinant DNA Research, Volume 12 
