A facile method for somatic, lifelong manipulation of multiple genes in the mouse liver.
Current techniques for the alteration of gene expression in the liver have a number of limitations, including the lack of stable somatic gene transfer and the technical challenges of germline transgenesis. Rapid and stable genetic engineering of the liver would allow systematic, in vivo testing of contributions by many genes to disease. After fumaryl acetoacetate hydrolase (Fah) gene transfer to hepatocytes, selective repopulation of the liver occurs in FAH-deficient mice. This genetic correction is readily mediated with transposons. Using this approach, we show that genes with biological utility can be linked to a selectable Fah transposon cassette. First, net conversion of Fah(-/-) liver tissue to transgenic tissue, and its outgrowth, was monitored by bioluminescence in vivo from a luciferase gene linked to the FAH gene. Second, coexpressed short hairpin RNAs (shRNAs) stably reduced target gene expression, indicating the potential for loss-of-function assays. Third, a mutant allele of human alpha1-antitrypsin (hAAT) was linked to Fah and resulted in protein inclusions within hepatocytes, which are the histopathological hallmark of hAAT deficiency disorder. Finally, oncogenes linked to Fah resulted in transformation of transduced hepatocytes.


CONCLUSIONS
Coexpression with FAH is an effective technique for lifelong expression of transgenes in adult hepatocytes with applicability to a wide variety of genetic studies in the liver.
Wangensteen
Kirk J
KJ
The Arnold and Mabel Beckman Center for Transposon Research, Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA.
Wilber
Andrew
A
Keng
Vincent W
VW
He
Zhiying
Z
Matise
Ilze
I
Wangensteen
Laura
L
Carson
Corey M
CM
Chen
Yixin
Y
Steer
Clifford J
CJ
McIvor
R Scott
RS
Largaespada
David A
DA
Wang
Xin
X
Ekker
Stephen C
SC
D019943:Amino Acid Substitution; D000818:Animals; D045744:Cell Line, Tumor; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D006867:Hydrolases / Q000172:deficiency / Q000235:genetics; D007414:Intestinal Neoplasms; D008099:Liver / Q000166:cytology / Q000473:pathology / Q000502:physiology*; D008113:Liver Neoplasms / Q000235:genetics; D008156:Luciferases / Q000235:genetics; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D010957:Plasmids; D002583:Uterine Cervical Neoplasms; D000515:alpha 1-Antitrypsin / Q000235:genetics
D018076:DNA, Complementary; D000515:alpha 1-Antitrypsin; D008156:Luciferases; D006867:Hydrolases; C020156:fumarylacetoacetase