Construction, electroporatic transfer and expression of ZpβypGH and ZpβrtGH in zebrafish
Tóm tắt
Recombinant transformation vectors (ZPβypGH and ZpβrtGH) consisting of fish growth hormone cDNA, and a reporter geneβ-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; 8 ohm resistance and 2 pulses) in the presence of one of these transformation vectors (100 μg 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 chosenF
0 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 chosenF
1 progenies was screened for germline transformation. Southern analysis of chosenF
1 progenies revealed the persistence of ZPβypGH or ZpβrtGH in 53% of theF
1 progenies. Southern analyses of chosenF
1 progenies and the frequency (53% ofF
1 ZpβrtGH and 53% ofF
1 ZP{β}ypGH) of transmission revealed the degree of mosaicism inF
0 transformants. Expression was confirmed from the 3–4 times elevated levels of activity of the reporter gene and 30–40% accelerated growth of transgenicF
0 andF
1 progenies.
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