Conditionally immortalized mouse hepatocytes for use in liver gene therapy.
OBJECTIVE
The use of hepatocytes for gene therapy is limited by the difficulty of maintaining and altering primary liver cells in culture. A conditionally immortalized mouse hepatocyte cell line has been developed which can be passaged indefinitely at the permissive temperature (33 degrees C), but fails to proliferate and dies at the non-permissive temperature (39 degrees C) in vitro.


METHODS
Hepatocytes were harvested from a 6 week-old male transgenic mouse ('immortomouse') carrying a thermolabile SV40 Large T gene, using a modified two-step collagenase perfusion method, and serially passaged at 33 degrees C for more than 1 year. To assess the ability of immortohepatocytes to engraft and populate mouse liver, cells were infused into partially hepatectomized congenic mice via the portal vein (n = 10) or the spleen with (n = 2) and without (n = 2) partial hepatectomy. The ability to transfect immortohepatocytes was assessed using the reporter gene enhanced green fluorescent protein (EGFP).


RESULTS
All immortohepatocytes in culture stained positive by immunohistochemistry for the hepatocyte markers albumin, AFP, CK8 and CK18. In early cultures a proportion of cells also stained strongly for the biliary epithelial markers CK7 and CK19. Late cell cultures were negative for M2PK and CK7 and stained variably with anti-CK19 antibodies. Cells transferred to the non-permissive temperature of 39 degrees C ceased proliferation and died within 1 week in vitro. Large T DNA was detected in the liver of all postoperative mice up to 2 weeks post-hepatocellular transplantation via PCR and Southern blot analysis. The immortohepatocytes were easily transfected with a reporter gene.


CONCLUSIONS
Immortohepatocytes can survive in vivo after transfer to liver, and will be useful as a model for hepatic gene therapy.
Allen
K J
KJ
The Murdoch Children's Research Institute, Royal Children's Hospital, Parkville, Victoria, Australia. allenk@cryptic.rch.unimelb.edu.au
Reyes
R
R
Demmler
K
K
Mercer
J F
JF
Williamson
R
R
Whitehead
R H
RH
D000818:Animals; D002460:Cell Line; D005260:Female; D015316:Genetic Therapy; D022781:Hepatocytes; D051379:Mice; D008822:Mice, Transgenic; D014162:Transfection