Genetic manipulation of rat hepatocytes in vivo. Implications for a therapy model of type-1 diabetes.
The successful expression of transferred insulin genes in non-pancreatic somatic cells could be an important step towards a somatic gene therapy of type-1 diabetes. Hepatocytes are potential target sites of a genetic manipulation. Using liposomes as a gene carrier system a method was established to manipulate rat hepatocytes in vivo. To prove this model, a bacterial marker gene (pRSVneo) was used which is normally absent in the mammalian genome. The genetic material was encapsulated into reverse-phase evaporated vesicles (REV). Using a sonication step for 60-90 s during vesicle preparation no significant loss of genetic information was observed. 70% of the liposomes were taken up by the liver within 10 min after i.v. application to male Wistar rats (1.5 mumol lipid and 10-15 micrograms DNA per 100 g b.w.). The transferred vector was detected in isolated nuclei of hepatocytes by Southern hybridisation up to 7 days. Integration events into the host genome were observed already 48 h after injection. The transcription of the transferred DNA (mRNA) as well as the translation (NPT-II activity) were demonstrated.
Leibiger
I
I
Institute of Biochemistry, School of Medicine, Ernst Moritz Arndt University, Greifswald, FRG.
Leibiger
B
B
Sarrach
D
D
Walther
R
R
Zühlke
H
H
D000818:Animals; D001345:Autoradiography; D015152:Blotting, Northern; D015139:Blotting, Southern; D004247:DNA / Q000235:genetics; D003922:Diabetes Mellitus, Type 1 / Q000235:genetics / Q000628:therapy*; D015316:Genetic Therapy; D008081:Liposomes; D008099:Liver / Q000166:cytology*; D008297:Male; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011919:Rats, Inbred Strains; D014158:Transcription, Genetic; D014162:Transfection; D014709:Vero Cells
D008081:Liposomes; D012333:RNA, Messenger; D004247:DNA