experiment completed, 1 x 10 7 macrophages transduced with the human GC gene were injected 
into the tail vein of a mouse. After two weeks, the mouse was sacrificed and various 
tissues were analyzed by PCR using amplimers that amplified a 192 bp fragment from the 
provirus beginning at the Nco I site and extending into the human GC cDNA. The results are 
shown in Figure 7. It can be seen that the bone marrow is positive for the human gene. 
C.6 Assay for the Presence of Helper Virus 
Viral supernatants from the psi-cre packaging line producing MFG-GC were negative for 
helper virus as assessed by the BAG mobilization assay, independently performed assays for 
helper virus performed by A.D. Miller were also negative for helper virus. The tissues of 
one of 4 control animals that were never exposed to the vector and 3 of 13 long term 
reconstituted animals resulted in the production of 1/2000 blue staining and Neo r 3T3 cell 
targets. This experiment was repeated twice with the same result. Each of the 13 animals 
studied carried 1 copy of the human gene in hematopoietic tissues and expressed GC activity 
several fold above the background. None of the controls were positive for MFG-GC by 
Southern analysis. The absence of helper virus in 10 animals that highly expressed the GC 
gene rules out the possibility that helper assisted reinfection was related to the 
expression of the GC gene. The presence of an agent in control animal tissues that could 
be detected by the BAG assay indicates that endogenous replication competent retroviruses 
were present in the mouse genome. These viruses are commonly found in mice and are known 
to be activated by radiation 186,871 . Kaleko et. al . , also have detected endogenous 
retroviruses in the course of gene transfer studies in mice that were unrelated to 
expression of the h-ADA gene in animals transplanted with retrovirally transduced bone 
marrow' 881 . 
C.7 Localization of the Human Gene Product in the Vesicular Compartment 
High resolution immunofluorescent and transmission EM ul trastructural studies were 
carried out by Dr. Simon Watkins in our university. These studies utilized the human 
specific monoclonal antibody 8E4. Control tissues from a normal mouse were completely 
negative. Tissues from animals reconstituted with bone marrow transduced by the MFG-GC 
vector showed many cells in which the human gene product could be identified, f igut^; 8 , 
is a high resolution immunofluorescent stain of the bone marrow localizing the human gene 
product in cellular vesicles. Conspicuously absent is any staining of the nucleus, figure 
I shows the presence of colloidal gold particles on the membranes of vesicles within the 
cell. The many positive figures present in these cells in vesicles, both perinuclear and 
beneath the plasma membrane, suggests that the cells are expressing the human GC gene 
product; further, that the enzyme is being distributed throughout the vesicular compartment, 
not only to lysosomes. These observations suggest that the enzyme is being secreted by 
these cells. We have demonstrated this fact in the media of cultured myoblasts transduced 
by the MFG-GC vector. 
SUMMARY OF DATA FROM MOUSE BONE MARROW TRANSPLANTS 
These data provide further evidence that retroviral vectors can efficiently transduce 
hematopoietic stem cells in the murine system. The presence of the human GC gene at copy 
numbers of 1-2 per mouse genome in primary CFU-S, in hematopoietic tissues of mice 
surviving up to 7 months after reconstitution, and in every spleen colony in secondary 
recipients demonstrates that primitive cells in the bone marrow were efficiently transduced 
and repopulated the tissues of the mice. It is unlikely that long lived committed 
progenitor cells were responsible for this effect considering the length of time after 
reconstitution. Comparisons to an earlier vector (N2-SV-GC) demonstrated a similar 
transduction efficiency in primary CFU-S and the tissues of long term reconstituted mice. 
This result indicates that high transduction efficiency is not a unique property of the MFG- 
GC vector per se, nor is it necessary in the murine system to develop a titer of greater 
than 10® particles/ml to achieve transduction efficiencies of hematopoietic stem cells that 
approach 100%. More importantly, this high efficiency transduction resulted from the bone 
Recombinant DNA Research, Volume 17 
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