Generation of pancreatic secretory trypsin inhibitor gene knockout mice

Generation of pancreatic secretory trypsin inhibitor gene knockout mice

International Congress Series 1255 (2003) 197 – 198 Generation of pancreatic secretory trypsin inhibitor gene knockout mice Masaki Omuraya a,b, Kaoru...

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International Congress Series 1255 (2003) 197 – 198

Generation of pancreatic secretory trypsin inhibitor gene knockout mice Masaki Omuraya a,b, Kaoru Hirokawa a, Masatake Araki c, Kimi Araki a, Masahiko Hirota b,*, Michio Ogawa b, Ken-ichi Yamamura a a

b

Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 860-8556, Japan Department of Surgery II, Kumamoto University Medical School, 1-1-1 Honjo, Kumamoto 860-8556, Japan c Gene Technology Center, Kumamoto University, Kumamoto 860-8556, Japan

Abstract Pancreatic secretory trypsin inhibitor (PSTI) is synthesized in the acinar cells of the pancreas and acts as a potent protease inhibitor that inhibits intrapancreatic activation of trypsin to prevent the occurrence of pancreatitis. Recently, we and other groups reported that some patients with chronic pancreatitis had a point mutation in the PSTI gene. Genetic mutations in the PSTI gene seem to promote a predisposition to pancreatitis, possibly by lowering the threshold of pancreatitis caused by other factors. In order to prove this hypothesis, we generated PSTI knockout mice using cre/mutant lox system. Heterozygous offsprings of chimeras appeared entirely normal. D 2003 Published by Elsevier Science B.V. Keywords: PSTI; Knockout mice; Pancreatitis; Trypsin; Protease inhibitor

1. Introduction Pancreatic secretory trypsin inhibitor (PSTI) is synthesized in the acinar cells of the pancreas and acts as a potent protease inhibitor that inhibits intrapancreatic activation of trypsin to prevent the occurrence of pancreatitis. Recently, we and other groups reported that some patients with chronic pancreatitis had a point mutation in the PSTI gene, and the mutation is considered to associate with the pathogenesis of chronic pancreatitis. Genetic mutations in the PSTI gene seem to promote a predisposition to pancreatitis, possibly by

* Corresponding author. Tel.: +81-96-373-5212; fax: +81-96-371-4378. E-mail address: [email protected] (M. Hirota). 0531-5131/03 D 2003 Published by Elsevier Science B.V. doi:10.1016/S0531-5131(03)00878-1

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M. Omuraya et al. / International Congress Series 1255 (2003) 197–198

lowering the threshold of pancreatitis caused by other factors. In order to prove this hypothesis, we are now generating PSTI knockout mice using cre/mutant lox system to introduce various point mutations as next step.

2. Materials and methods Genomic DNA corresponding to the PSTI gene was isolated from a B6 genomic library. A lox71-PGK-neo-lox2272 cassette was inserted into the BglII site positioned before the initiation codon. The linearized targeting vector was introduced into TT2 embryonic stem cells through electroporation. Chimeras were generated by aggregation with ICR morula embryos.

3. Results Three clones out of 335 G418 resistant ES clones were identified to have undergone homologous recombination by polymerase chain reaction and Southern blot analysis. Two of the three clones were successfully introduced into the germ line of mice.

4. Conclusion We have obtained three cell lines arising from independent targeting events and established two mice lines. Heterozygous offsprings of chimeras appeared entirely normal.