have a sensitivity of approximately one proviral copy per 10-20 cells. The 
former method is more informative since gross deletions and rearrangements can 
be detected. A new method for amplifying a specific DNA sequence. PCR 
(polymerase-catalyzed reaction), has been developed (Erhlich et al.. 1986) 
which increases sensitivity of the probe methods to 1 gene copy per 10^ to 10^ 
cel 1 s. 
2. RNA vector transcripts can be demonstrated by RNA dot blots. Northern 
analysis, nuclease protection assays, solution hybridization, and in situ 
hybridization. 
3. Expression of a selectable marker can be demonstrated by metabolic 
resistance to toxic agents or by assay of enzyme activity of the transferred 
D 
gene. When the transferred genes are ADA and Neo (as in SAX), then specific 
methods include (a) growth of colony forming units (CFU) in the presence of the 
toxic neomycin analogue G418, (b) assay of neomycin phosphotransferase (NPT) 
activity, c) assay of ADA activity by isoenzyme analysis, and (d) growth of 
D 
lymphocytes under conditions selective for the expression of either the Neo or 
the ADA gene. 
4. Rescue of infectious retroviral vectors from treated cells by 
incubation in the presence of helper virus. 
I. Studies in Mice; 
A. Efficiency : 
Literature review: Utilizing a number of Moloney murine leukemia 
virus (Mo-MuLV) or Moloney murine sarcoma virus (Mo-MSV) -based vectors, other 
investigators have found a 10-20? rate of vector gene transfer into murine 
hematopoietic cells (Wilkins et al.. 1984; Joyner et al.. 1983; Miller et al . 
1984 and Table la). By employing a number of manipulations (either singly or in 
combination), including pretreatment of mice with 5-fl uorouracil (5-FU). 
[24] Recombinant DNA Research, Volume 12 
