The utility of retroviral vectors is often limited by the low and variable 
titer of the recombinant viruses and the low efficiency of expression of the 
transduced gene in the infected cell. In the studies reported here, two 
different designs of retroviral vectors are examined and the effect of internal 
viral sequences on the function of each is investigated. 
Double expression (DE) vectors The structure of two DE vectors are shown in 
Fig. 1 A . In this type of vector first described by Cepko et.al.(2) The 
selectable Neo gene replaces the viral gag/pol coding sequences and is expressed 
from an unspliced RNA species. The ADA cDNA replaces the sequences coding for 
the viral envelope gene and is expressed from a sliced RNA species. In DE-NA/S, 
similar but not identical to the vectors described by Cepko et. al.(2), the DNA 
fragment derived from the M-MuLV intron (which encodes the 3' splice junction 
sequences) is about 800 base pairs (bp) long . In DE-NA/X, the viral DNA 
fragment encoding to 3' splice junction is about 4000 bp long which includes a 
large portion of the retroviral intron. Two vector specific transcripts are 
present in NIH 3T3 cells infected with DE-NA/S and DE-NA/X virus as shown in 
Fig. 2 A. The slower migrating RNA species corresponds in size to the unspliced 
RNA form which is longer in DE-NA/X compared to DE-NA/S infected cells, 
reflecting the presence of additional intron sequences in DE-NA/X. The faster 
migrating RNA species expressed from each vector corresponds in size to the 
spliced RNA form and as 
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Recombinant DNA Research, Volume 12 
