Construction, electroporatic transfer and expression of ZpfJypGH and ZpfJrtGH in zebrafish.

ABSTRACT

Recombinant transformation vectors (ZPPypGH and ZpprtGH) consisting of fish growth hormone cDNA, and a reporter gene p-galactosidase driven by fish promoter (Zp) were constructed. Freshly fertilized eggs of zebrafish (Brachydanio rerio) were electroporated at optimum conditions (0.07 kV voltage; 25 J.1F capacitance; 00 ohm resistance and 2 pulses) in the presence of one of these transformation vectors (100 J.1g circular DNNml). In either cases 72% of the electroporated eggs successfully hatched, in comparison to the 85% hatchability of the control eggs. Genomic DNA extracted from fins of randomly chosen Fo individuals was screened (by Southern blot hybridization); the transgenes were retained in the host-. genome of all the randomly chosen adult transformants. Fin-positive presumptive founder parents were crossed with control counterparts and the DNA of randomly chosen F] progenies was screened for germ-line transformation. Southern analysis of chosen F1 progenies revealed the persistence of ZPPypGH or ZpPrtGH in 53% of the F1 progenies. Southern analyses of chosen F1 progenies and the frequency (53% of F1 ZpPrtGH and 53% of F1 ZPPypGH) of transmission revealed the degree of mosaicism i!1 F 0 transformants. Expression was confirmed from the 3-4 times elevated levels of activity of the reporter gene and 30-40% accelerated growth of transgenic Fo and F1 progenies.