viral long terminal repeat (LTR) sequences or by an exogenous internal promoter 
derived from another gene. The degree of expression may also vary from 
cell-to-cell depending on the site of integration, the extent of 
differentiation of the target cell and the regulatory sequences which are a 
component of the retroviral construct. 
In vitro studies 
Gene transfer and expression in. vitro has been achieved in transformed 
cell lines, murine and human hematopoietic cells and nontransformed human 
diploid fibroblasts (summarized in Table 2. Appendix A). One of the first 
successful attempts at retroviral gene transfer involved the insertion and 
expression of a cDNA for the human hypoxanthine phosphoribosyl transferase 
(HPRT) gene into HPRT-deficient rat 208F cells. SV40-transformed Lesch-Nyhan 
(LNSV) human fibroblasts, and Epstein-Barr virus-transformed Lesch-Nyhan human 
lymphoblasts (Miller et al.. 1984). Other early investigations of retroviral 
D 
gene transfer into cultured cells have utilized the bacterial Neo gene which 
confers resistance to the neomycin analogue. G418, in eucaryotic cells. The 
D 
Neo gene has been inserted into a retroviral vector alone, as in the case of 
MLV-NEOI and N2. or in combination with other genes in double expression 
vectors to be used as a dominant selectable marker in expressing cells. The 
D 
MLV-NEOI vector developed by Joyner et al., containing the Neo gene under 
transcriptional control of the SV40 early promoter, was used to infect mouse 
bone marrow cells in vitro . When hematopoietic precursor cells were analyzed 
for expression of the Neo R gene, 0.32 of the CFU-GM from a population of cells 
infected with MLV-NEOI were resistant to concentrations of G418 normally toxic 
to uninfected marrow cells. Neo R gene specific RNA was also detected by RNA 
dot blot hybridization of labelled Neo R sequences to RNA extracted from 
G418-resistant colonies. This finding was important because it demonstrated 
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Recombinant DNA Research, Volume 12 
