B. 3 Isolation of Virus Producing Cells and Titering 
In all of the following studies, we made comparisons between the MFG-GC and N2-SV-GC 
vectors. The MFG-GC construct was co-transfected with pSV 2 Neo to the ecotropic packaging 
line, psi-cre, using calcium-phosphate precipitation. The molecular ratio of MFG-GC and 
pSV 2 Neo was 20:1. After G418 selection (400ug/ml), 30 clones were isolated and grown up to 
near confluency in 100mm dishes. The culture medium of these clones was used as the virus 
source. As a target cell for titering the virus, NIH 3T3 cells were used. The virus 
containing medium was added to NIH 3T3 cells plated at a density of lxlO 5 cells per well in 
six-well dishes. Polybrene was added to the medium at concentration of 8ug/ml to facilitate 
uptake of the virus by cells. After 2 hour incubation at 37°C, the virus containing medium 
was removed and new medium was added. Following 48 hours incubation at 37°C, the cells were 
harvested and assayed for enzymatic activity, Western blot and Southern blot. The titer 
of lxlO 6 cfu/ml for the psi-cre producer of N2-SV-GC was based on the number of G418 
resistant colonies of infected NIH 3T3 cells. The titer of the MFG-GC vector was estimated 
by Southern blots of MFG-GC infected 3T3 cells and was compared to Southern blots of non- 
! selected 3T3 cells infected by the N2-SV-GC vector. The MFG-GC viral producer line clone 
(GC-psi-cre #4) with a titer of 1 x 10 6 was chosen for use in these experiments. 
C. EX VIVO GENE TRANSFER AND SYNGENEIC BONE MARROW TRANSPLANTATION IN MICE 
C. 1 Gene Transfer: Mouse Hematopoietic Cells 
For these studies, we followed a protocol essentially as outlined by Bodine et al <40) . 
Bone marrow was obtained from animals treated for 3-5 days with 5-FU, precultured for 2 days 
with poke weed mitogen spleen-cell -conditioned medium and WEHI 3/b cell conditioned medium 
1 (CM), then co-cultured for 2 days with clones of VPL producing titers of 5 x 10 5 to 10 6 for 
either MFG-GC or N2-SV-GC vectors. Donor mice were male C57BL6/J-GPI 8 which have a 
I glucophosphate isomerase (GPI) isozyme designated as GPI-1". This serves as a convenient 
marker of donor cells. These donor mice also have another genetic marker known as 
hemoglobin "diffuse" (Hbb d /Hbb d ). The recipient mice were young female C57BL6/J-GPI b mice 
that have a GPI - l b isoenzyme. The GPI markers permitted the estimation of the % of donor 
cells after transplantation. 
C.2 Transduction of CFU-S 
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I 
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i 
i 
The efficiency of transduction and expression of the GC gene was estimated by 
analyzing individual colonies harvested from the spleen of lethally irradiated mice at 12 
days after BMT. Eighty six (86) colonies from the spleens of mice given MFG-GC transduced 
cells and thirteen (13) colonies from mice reconstituted with marrow infected with N2-SV-GC 
were analyzed for comparison. Colonies from lethally irradiated mice transplanted with 
normal syngeneic marrow were used as controls. Representative data are shown in Figure 3. 
Although the Southern blot revealed that all the colonies in spleen from both groups of mice 
car r i ed approx i matel y the same number of transferred human GC genes (1-2 copies/genome) 
(figure 3C), the colonies from MFG-GC mice had enzymatic activities that ranged from 2-5 
fold above the control s , whereas the colonies from N2-SV-GC mice were not greater than 
controls (Ifig^e^SA) . Western blot results were congruous with the enzymatic activity 
results i.e. very Tittle human enzyme protein could be detected in colonies derived from 
N2-SV-GC infected marrow (Figure 38) . 
C.3 Long Term Reconstituted Mice 
By two months after BMT in mice given 2 x 10 6 bone marrow cells, circulating white 
blood cells were >90% donor type as assessed by GPI, and remained >90% until sacrifice. 
In addition, the majority of circulating white blood cells were positive for the human gene 
product by immunocytochemical analysis. Animals were sacrificed between four and seven 
months after BMT. Various tissues from mice given MFG-GC infected cells were analyzed by 
enzymatic activity, Western blot, and Southern blot analyses. The enzymatic activity data 
in lllillll summarizes the measurements made on 10 animal s reconstituted with MFG-GC 
Recombinant DNA Research, Volume 17 
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