Expression of the human insulin gene in the gastric G cells of transgenic mice.
The goal of this study was to engineer gastrin-producing G cells of the gastric antrum to produce insulin. A pGas-Ins chimeric gene in which the gastrin promoter drives expression of the human insulin gene was constructed and was validated by transient transfection of GH4 and AGS cells. RT-PCR analysis and sequencing revealed three forms of differentially spliced insulin mRNA in GH4 cells transiently transfected by pGas-Ins. Gas-Ins transgenic mice were generated utilizing this chimeric gene. Northern blot analysis, in situ hybridization, and immunohistochemistry demonstrated expression of the human insulin gene specifically in antral G cells. Northern blot analysis demonstrated that the shortest of the insulin mRNA three forms is predominantly expressed in stomach tissue. RT-PCR analysis also showed expression of the transgene in colon, pancreas, and brain tissues that was undetectable by northern analysis. We conclude that gastrin promoter can be used for targeting expression of human insulin to antral G cells and that antral G cells can express human insulin. Further refining of the chimeric gene design is required to enhance expression.
Zhukova
E
E
CURE: Digestive Diseases Research Center, UCLA School of Medicine, Los Angeles, CA 90095, USA. ezhukova@ucla.edu
Afshar
A
A
Ko
J
J
Popper
P
P
Pham
T
T
Sternini
C
C
Walsh
J H
JH
D000818:Animals; D015152:Blotting, Northern; D002478:Cells, Cultured; D002678:Chimera; D017931:DNA Primers / Q000737:chemistry; D005260:Female; D019863:Gastrin-Secreting Cells / Q000378:metabolism*; D005755:Gastrins / Q000096:biosynthesis; D015870:Gene Expression; D006801:Humans; D007124:Immunoenzyme Techniques; D017403:In Situ Hybridization; D007328:Insulin / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D016133:Polymerase Chain Reaction; D011706:Pyloric Antrum / Q000166:cytology / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D005755:Gastrins; D007328:Insulin; D012333:RNA, Messenger