HEPATOCELLULAR TRANSPLANTATION AND TARGETING GENETIC MARKERS TO HEPATIC CELLS 
to an order of magnitude) by performing multiplex analysis with other templates at a 
similar initial concentration (Wang, 1990), This method should be able to determine the 
order of magnitude of transplanted cells ranging down to 0.01% of all cells in the 
sample . 
The least useful methods are those which involve detection of the NEO-R protein 
which are dependent upon gene expression and exhibit relatively poor sensitivity. These 
methods include enz)n3iatic assay for NEO-R and immunohistochemical methods which are 
inadequate with currently available antibodies. If improved reagents become available, 
this method would provide for a quantitative assessment of the number of transduced 
cells and analysis of their location, morphology, and function. 
The detection of proviral expression by in situ hybridization would be an ideal 
method for detecting transduced and transplanted cells and would enable coincident 
studies of morphology and tissue specific gene expression in the transduced cells. This 
method is often considered inadequate to detect 10'*-10'® cells due to the relatively poor 
signal to noise ratio. Dr. Dorothy Lewis (Department of Immunology, BCM) has recently 
developed a technique for detecting HIV infected cells in solid organs (especially 
placenta and liver) using in situ hybridization and confocal laser scanning microscopy 
for greater resolution (Lewis et al, 1990). Confocal microscopy increases sensitivity 
by eliminating the background which is evenly distributed through the photo -emulsions 
and focusing on layers immediately adjacent to cells where the desired signal is 
concentrated. We will be working with Dr. Dorothy Lewis to apply these methods for the 
detection of the LNL6 expression by in situ hybridization. 
The presence and expression of the NEO-R gene could also allow pharmacological 
selection of transduced cells if there is an opportunity to prepare hepatocytes from the 
host. This will be possible if the intact organ is recovered during subsequent OLT, 
Hepatocytes will be isolated and cultured +/- G418 using methods previously described 
to select NEO-R hepatocytes (Ledley et al, 1987). This would provide sensitive 
detection of transduced cells and a quantitative assessment of the fraction of cells 
containing the selectable marker. 
4. Autopsy. The informed consent will indicate the importance of obtaining an 
autopsy if the patient dies after HCT, and every attempt will be made to obtain 
permission for an autopsy at the time of death. If a complete autopsy is not permitted, 
we will ask for permission to retrieve a portion of the transplanted liver. At autopsy, 
portions of the transplanted liver and spleen will be studied for detectable evidence 
of the integrated or expressed provirus as described in the previous section. 
Conventional histological examination will be performed of all available organs with 
particular attention to any evidence for embolization resulting from the cellular 
transplant procedure, autoimmune phenomenon, dysplastic cells, or other untoward 
phenomenon . 
5. Blood samples from the donor and recipient will be analyzed for the presence 
of polymorphic proteins (Ritzman and Johnson, 1983). In theory polymorphic variation 
in serum proteins is easily detected by standard clinical tests such protein 
Recombinant DNA Research, Volume 14 
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