Abstract
Retroviral gene transfer is most convenient for permanently expressing introduced genes but is hampered by the requirement for DNA synthesis. We reasoned that a producer cell reservoir in the spleen should release retrovirus particles into the portal circulation with the producer cell survival controlled by the natural process of allograft rejection. To test our hypothesis, we used NIH 3T3 cells producing an ecotropic ψCRE-nls-lacZ retrovirus containing E. coli lacZ. Typically, the retrovirus led to X-gal stainable lacZ expression in 60%-90% NIH 3T3 cells. To test in vivo gene transfer, we allografted 1×106 producer cells by intrasplenic injection 24 hrs after a 35% hepatectomy into C57BL/6J mice. In situ hybridization shows H3 histone mRNA expression, indicating DNA synthesis, in ≈4% of the hepatocytes at 48 hrs after 35% partial hepatectomy (Vemuru et al., Hepatology 1992; 16:968). At 5 days after cell transplantation, lacZ activity was observed in hepatocytes, as well as transplanted producer cells, which were visible within or adjacent to portal areas in the liver. Colocalization of lacZ activity and characteristic peroxisomes by histochemistry allowed a distinction between hepatocytes and producer cells. LacZ activity was observed in only hepatocytes during the 8 week duration of the study. Surveys of organs for retroviral gene transfer by PCR demonstrated lacZ DNA in the liver, spleen, testis, lungs and small intestine and not in the brain, kidney, heart or skeletal muscle. The overall magnitude of hepatic gene transfer approached <1% hepatocytes. To determine whether growth factor infusion could potentially replace partial hepatectomy, we next examined gene transfer in allogeneic MT42 TGF-a transgenic mice, which exhibit a high baseline level of DNA synthesis. Transplantation of producer cells into the transgenic mice without prior hepatectomy also resulted in lacZ positive hepatocytes. CONCLUSIONS: A simple and convenient system for retroviral gene transfer into the liver of intact animals was developed. Such a system shall facilitate a variety of experimental studies in animals to address cell lineage analysis, as well as expression and regulation of therapeutic genes.