infected bone marrow and 2 animals reconstituted with N2-SV-GC infected bone marrow. I e 
Representative Southern analyses from three MFG-GC animals are presented in Figure 4B. 
Completely similar data were obtained on Southern blots of tissues from 22 animals studied 
with the MFG-GC vector. Southern blots of N2-SV-GC animals are shown in Figure 4C. 
Southern blots were positive for the human gene in all of the individual tissues. The 
enzymatic activity of hematopoietic tissues (bone marrow, spleen, thymus and lymph node) 
from mice reconstituted with MFG-GC infected marrow exceeded the activity of control tissues 
in all but a few specimens of lymph node and thymus. This most likely reflects the failure 
to obtain adequate thymus and lymph node tissue from irradiated animals. The average li 
increase in GC activity was 3 fold in spleen and 6 fold in bone marrow. By comparison, the l> 
hematopoietic tissues from mice given N2-SV-GC infected BM cells showed little or no « 
increase above control levels of activity. From the Southern blot analyses of hematopoietic r 
tissues, it can be concluded that the MFG-GC vector was efficient in infecting early stem t 
cells capable of long term reconstitution of bone marrow. The copy number was approximately s 
1-2/mouse genome. The data accumulated on non-hematopoietic tissues (liver, lung) was also j $i 
informative. These tissues normally are supplied with bone marrow derived cells on a Si 
continuing basis. Under normal physiologic circumstances, bone marrow derived cells in | 51 
these tissues are primarily macrophages. In liver, tissue macrophages (Kupffer cells) e; 
constitute approximately 15% of the organ 183,841 . If all of the liver macrophages were Ik 
replaced in MFG-GC reconstituted animals by the progeny of transduced hematopoietic stem w 
cells derived from the bone marrow, the copy number of the vector in the liver should be ti 
about 0.15/cell. The results of Southern blot analyses demonstrated that the MFG-GC vector n 
resulted in a copy number in liver of approximately 0.1/cell. This is consistent with a ei 
high transduction efficiency of HSC by the MFG-GC vector and the ability of HSC to 1 
repopulate the macrophage lineage. 
C.4 Secondary BMT 
As a further measure of the ability of the MFG-GC vector to transduce self renewing 
pluripotent HSC, we performed secondary bone marrow transplantations using bone marrow 
collected from three long term reconstituted mice. These secondary transplant recipients 
were sacrificed at twelve days and spleen colonies were harvested. All of the 27 secondary 
colonies were positive for the human GC gene by Southern blot analyses (Figure 5A) . The 
enzymatic activities of the 27 colonies were 2-7 times higher than that of control CFU-S 
(Figure 5B) . These data provide further evidence of very efficient transduction of HSC by 
the bone marrow infection protocol and sustained high levels of expression of human GC in 
the progeny of HSC responsible for forming spleen colonies in secondary recipients. 
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C.5 Expression of Human GC in Differentiated Macrophages 
Culture of BM with M-CSF resulted in nearly pure colonies of macrophages that were 
actively phagocytic as evidenced by the ability to take up latex beads (Figure 6A,B). These 
cultures represented approximately a five log expansion of BM precursors. The enzymatic 
activity of the macrophages grown from BM of eight long term reconstituted mice was on 
average 4 fold greater than that of control macrophages (Figure 6 C). Western blots of 
macrophage extracts showed an intense band of human GC protein not present in control cells 
cultured from normal mouse bone marrow. The copy number in these cells was approximately 
1-2/cell by Southern blot analyses (Figure 6D) . Immunochemical staining of these cultured 
macrophages for the human gene product revealed that most of the cells exhibited positive 
staining, whereas there was no staining in control cultures (.Figure - :;6AyB) . Macrophages 
cultured from the bone marrow of long term reconstituted mice that were positive for the 
expression of the human GC gene were studied in transplantation studies. The purpose of 
this investigation was to determine the distribution and fate of macrophages in a syngeneic 
recipient. Reports published some years ago suggested that peritoneal macrophages were 
distributed widely, but little data was available on how long these cells persisted in the 
host 1851 . The availability of mouse macrophages transduced with the human gene provided 
marked cells which could be traced by immunocytochemical and PCR methods. In the first 
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Recombinant DNA Research, Volume 17 
