Insertion of a functional copy of six2 to generate a transgenic mouse via piggybac transposase

Abstract

Identification of gene function based on known mutations remains an integral objective in the field of basic science. The adult Br heterozygous mouse displays a reduced number of nephrons in association with chronic renal failure, hypertension and reduced Six2 expression during embryonic renal morphogenesis. The purpose of this study was to determine whether a functional copy of Six2 could be inserted into the Br mouse genome in an effort to overexpress Six2. After processing and isolation by restriction enzyme digestion and pulse field gel electrophoresis, 26kb Six2 BAC DNA fragment was then cloned into the mGENIE-3-BAC transposon vector via the in vitro Gibson Assembly method. The mGENIE-3-BAC-Six2 construct was subsequently confirmed by colony PCR, restriction enzyme digestion; the construct was also tested for functionality and expression in human cell lines and transgenic mice. Our data indicates that our single plasmid transposase-mediated approach to transgenesis requires fewer embryos, while capable of incorporating 42kb of exogenous DNA into the mouse genome.