Construction, electroporatic transfer and expression of ZpβypGH and ZpβrtGH in zebrafish

Abstract

Recombinant transformation vectors (ZPβypGH and ZpβrtGH) consisting of fish growth hormone cDNA, and a reporter gene β-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 μF capacitance; ∞ ohm resistance and 2 pulses) in the presence of one of these transformation vectors (100 μg circular DNA/ml). 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 F₀ 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 F₁ progenies was screened for germline transformation. Southern analysis of chosen F₁ progenies revealed the persistence of ZPβypGH or ZpβrtGH in 53% of the F₁ progenies. Southern analyses of chosen F₁ progenies and the frequency (53% of F₁ ZpβrtGH and 53% of F₁ ZP{β}ypGH) of transmission revealed the degree of mosaicism in F₀ transformants. Expression was confirmed from the 3-4 times elevated levels of activity of the reporter gene and 30-40% accelerated growth of transgenic F₀ and F₁ progenies.