Transmission and expression of an integrated reporter construct in three generations of transgenic mud loach (Misgurnus mizolepis)

Abstract We produced transgenic mud loach lines by microinjecting a CAT reporter gene driven by the carp β-actin promoter into fertilized eggs and demonstrated successful integration, transmission to F 3 , with variable patterns of expression in different tissues. Microinjection yielded transgenic F 0 individuals with a frequency that ranged from 9.3 to 27.8% among microinjected groups. Of 63 transgenic founders identified, seven males and one female were found to transmit the transgene to F 1 offsprings. All founders were mosaic in their germ cells; consequently the transmission frequency ranged from 3.7 to 33.4%. Most, but not all, F 1 and F 2 transgenics transmitted the transgenic construct to F 2 and F 3 progeny, respectively, with expected Mendelian ratios. Immunohistochemical in situ analysis showed that the transgene pFV4bCAT was effectively expressed in transgenic germlines. Reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbant assay (ELISA) revealed that the presence of CAT mRNA and the expression patterns in different tissues of adult individuals varied among each transgenic line; the highest level of CAT expression was observed in muscle from a transgenic F 1 line, corresponding to more than 89-fold compared to the basal control level.