HEPATOCELLULAR TRANSPLANTATION AND TARGETING GENETIC MARKERS TO HEPATIC CELLS 
(Gilliand et al, 1990; Wang and Mark, 1990). Control PCR reactions are routinely 
performed with all reagents in the absence of template to assess potential 
contaminations . 
The marker rescue assay is performed using an NIH3T3 cell line which contains 
provirus with an E. coli E-galactosidase gene in a packaging defective construct. This 
cell line does not produce retroviral particles since no gag/pol/env sequences are 
present. If this cell is superinfected with a virus containing these determinants 
(helper virus), a E-gal retrovirus is produced which can be titered on NIH3T3 cells. 
This cell line will be used to assay for the presence of infectious, helper virus in 
media and patient samples. This method is very sensitive since infectious particles are 
propagated throughout the culture. Retrovirus particles can also be assayed by the 
presence of reverse transcriptase as described (Goff et al, 1981). 
E. Evalviation of engraftment. 
Estimation of the number of cells in the transplanted liver. We estimate that the 
volume of liver cells infused represents 1-7% of the number of hepatocytes in a normal 
liver. All of the transplanted cells will be exposed to the vector and we expect 5- 
20% of these cells to be transduced. In animal experiments, 50% of transplanted cells 
have survived. The fraction of cells containing the provirus may be calculated as the 
fraction of cells transplanted and the transduction efficiency: 
Table 8. Fraction of cells which will contain marker gene as a function of the 
fraction of host hepatic cells transplanted and efficiency of transduction. 
<-■ 
% cells 
efficiency of transduction 
0.05 0.1 0.2 
---> 
0.007 
3.50E-04 
7.00E-04 
1.40E-03 
0.024 
1.20E-03 
2.40E-03 
4.80E-03 
0.07 
3.50E-03 
7.00E-03 
1.40E-02 
All of these values are within the range of detection of provirus by PCR. These 
values are also within the range of detection of HIV expression by in situ hybridization 
using confocal microscopy (Lewis et al, 1990). If there is proliferation of the 
transplanted cells relative to the host cells , the fraction of cells containing the 
provirus may be further increased. 
Methods for assessing engraftment. As described above, the limitation of most 
previous animal experiments with HCT has been failure to identify cells in the 
recipient. We believe this is a difficult problem and we will employ a variety of 
different approaches in an effort to optimize the probability of success. The purpose 
of proposing genetic marking of the hepatocytes is to add this method to the available 
approaches . 
Recombinant DNA Research, Volume 14 
[813] 
