HEPATOCELLULAR TRANSPLANTATION AND TARGETING GENETIC MARKERS TO HEPATIC CELLS 
the PAH apoenzyme was expressed in transduced cells (measured in vitro with exogenous |^| 
cofactor and reducing agents) , no holoenzyme was present in NIH3T3 cells and they | 
remained unable to grow in tyrosine free media or to metabolize [^''C] -phenylalanine to 
[‘‘‘C] -tyrosine in culture (Ledley et al, 1986; 1987b). Supplementation of NIH3T3 culture 
media with pharmacological concentrations of the cofactor, dithiothreotol , and catalase i 
constituted the holoenzyme in these cells (Ledley et al , 1987b). In contrast, ji 
transduction of the murine hepatoma cell line, hepala, which does not constitutively 
express PAH, constituted the holoenzyme and enabled these cells to grow in the absence j 
of tyrosine and metabolize [^^C]-phe in culture (Ledley et al, 1986). 
In order to evaluate the potential for transducing primary hepatocytes using ■ 
retroviral vectors, hepatocytes were harvested from murine or human livers by 
collagenase digestion (Amsterdam and Jamieson, 1972; 1974); cultivated in hormonally ; 
defined (Darlington et al, 1987) tyrosine free media which selects hepatocytes from | 
fibroblasts or other cells in the hepatic parenchyma (Ledley et al , 1987a; Peng et al, ! 
1988) ; and transduced with a variety of vectors containing the NEO-R gene under the ^ 
transcriptional control of different promoters. Retroviral transduction was assessed p 
by scoring G418 (a neomycin analogue) resistant clusters. The MoTN vector (from Dr. 
Alan Bernstein) containing the herpes virus TK promoter transduced hepatocytes at high [I 
efficiency, while equivalent titers of vector using the LTR promoter (ZIPNEO-SV(X) from b 
D r. Richard Mulligan) transduced hepatocytes at lower efficiency, and a vector using the t 
SV40 promoter (MLVNeo.l) produced only rare G418 resistant cells (Ledley et al , 1987a). ■ 
These results paralleled the ability of these same promoters to direct hepatic 't' 
expression in transgenic animals (Stewart et al , 1987) and suggested that the - 
transduction efficiency reflected the activity of these promoters in hepatocytes (Ledley 
et al , 1987a). Subsequent studies by Wilson et al (1988a), using a £-galactosidase (E- 
gal) reporter gene, and ourselves (Peng et al, 1988), using the N2 vector, have found i 
high-level expression from the retroviral LTR. Hepatocytes transduced with recombinant i 
retroviral vectors have continued to exhibit characteristic morphology in culture and i' 
to express liver specific functions (phenylalanine hydroxylase, alpha, -antitrypsin) ■' 
(Ledley et al, 1987a). ! 
In order to optimize gene expression in hepatocytes, a vector (NASPAH) was 
constructed in which the AAT enhancer (Shen et al, 1987) and SV40 promoter elements were 
used to drive transcription of a PAH cDNA within the vector N2 (Peng et al, 1988). ' 
Transcription from the integrated provirus was assayed in NIH3T3 cells and primary lOj 
hepatocytes selected for G418 resistance. G418 resistant colonies were obtained in 1<| 
both NIH3T3 cells and primary hepatocytes indicating that the retroviral LTR was active |(j) 
in both cells. The internal AAT promoter retained its tissue specificity and drove (i| 
transcription in hepatocytes but not in NIH3T3 cells (Peng et al, 1988). These data 
illustrate the complexity of gene regulation within the provirus since the two | 
nonspecific LTR enhancers might be expected to activate the SV40 promoter in NIH3T3 
cells. This complexity suggested that provirus or AAT sequences alter the cis enhancer ki 
effects of the LTR on the SV40 promoter (Peng et al, 1988). |jl 
[ 792 ] 
Recombinant DNA Research, Volume 14 
