Generation of transgenic homozygous line carrying the CAT gene in mud loach Misgurnus mizolepis

Abstract

SUMMARY: A stable homozygous line of transgenic mud loach (Misgurnus mizolepis) carrying a CAT reporter construct was developed. The founder transgenic mud loach was produced by microinjection of a CAT reporter gene driven by a carp beta-actin promoter and regulatory region (pFV4CAT) into fertilized eggs. The presumptive homozygous transgenic fish were generated by sister–brother mating between two heterozygous transgenic F2 siblings. Out of 28 transgenic individuals tested, five fish transferred the transgene into their progeny at a rate of 100%, four fish at 80–91%, and remaining 19 individuals inherited transgenic construct at a rate of approximately 50%. The level of expressed CAT enzyme in tissues (liver and spleen) from homozygous transgenic fish was higher than in those from heterozygous transgenic fish, although the increased amounts of CAT expression were slightly variable among homozygous individuals. The average level of CAT expression in homozygous fish was 1.8-fold (for liver) and 1.6-fold (for spleen), that of heterozygous fish.