B. Vector mutation and stability 
(1) Mutation of inserted vector genome. 
What is the evidence that the SAX vector that was constructed and 
sequenced will be the same as the vector that is inserted into the target cell? 
After Integration, how stable will the vector be with time? Without sequencing 
a large number of recipients we cannot answer these questions, but the 
literature allows us to make some general statements about the accuracy and 
stability of gene transfer. For organizational purposes we will trace the 
steps from vector construction to Infection and discuss the chances for 
mutation at each step. 
(a) Transfection. Production of the helper-free producer line is 
accomplished by transfection, a process with a high frequency of mutations. 
One percent of SV40-based recombinant shuttle vectors are defective after 
transfection by the DEAE-dextran method or by protoplast fusion (Razzaque et 
al.. 1983). Using a similar system. Calos et al . (1983) found a large number 
of base substitutions In addition to deletions, duplications and other 
rearrangements resulting In an estimated mutation frequency of one to several 
percent. Subsequent studies by this group demonstrated a similar frequency of 
mutation using the calcium phosphate method of transfection in monkey and mouse 
cell lines, but found the mutation frequency to be only 0.1% in two human cell 
lines studied (Lebkowski et al.. 1984). 
The magnitude of mutations from transfection of the SAX vector are 
minimized by selecting clones for neomycin resistance and then requiring that 
they also produce human ADA. Both require that the internal structure of the 
genome be largely Intact. By selecting cells that meet these requirements, we 
are excluding major mutations. If subtle changes affecting expression or 
pathogenicity are present, they can only be ascertained by in vivo testing. 
Recombinant DNA Research, Volume 12 
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