The delivery system can be very efficient. Up to 1002 of tissue culture 
cells may be infected by retroviral vectors. Observed efficiencies of N2/SAX 
gene transfer into primary hematopoietic cells are as follows: (a) Mice: 
50-802: (b) Sheep: 10-302: (c) Dogs: 3-52: (d) Monkeys: 10-252; (e) 
Human: 1-42. The values for the sheep are known to be over-estimates of the 
true level of infection based on DNA analysis of the cells. It is likely that 
some mechanism artificially elevates the observed number of G418-resistant 
colonies. For example, positive clones may release the Neo R phosphotransferase 
enzyme which then can diffuse out to protect nearby surrounding colonies. The 
actual percentage of vector-containing sheep CFU-C is probably one order of 
magnitude lower than listed above. 
(3) How Is the structure of the added DNA sequences monitored and 
what is the sensitivity of the analysis? Is the added DNA 
extrachroDOSonal or Integrated? Is the added DNA unrearranged? 
The structure of vectors Integrated Into cellular DNA Is monitored 
utilizing Southern hybridization techniques. Routinely this method will detect 
about 1 vector copy per 10 to 20 cells. However, using the recently developed 
technique of PCR (polymerase-catalyzed reaction), a procedure which amplifies a 
c c 
specific DNA sequence. 1 vector copy per 10-10 cells can be detected. 
Animal studies indicate that cells contain the retroviral vectors integrated 
Into chromosomal DNA in an unrearranged form. Studies in tissue culture have 
demonstrated Instances of vector rearrangement. These are discussed in Section 
I.B.2.C. 
(4) How many copies are present per cell? How stable Is the added 
DNA both In terns of Its continued presence and Its structural 
stab 11 Ity? 
Studies i_n vitro Indicate that usually only one copy of vector DNA is 
present per cell. However, experimental conditions can be arranged so that 
there are multiple copies per cell. 
Recombinant DNA Research, Volume 12 
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