Electroporatic transfer, stable integration, expression and transmission of pZpβypGH and pZpβrtGH in Indian catfish, Heteropneustes fossilis (Bloch)

Abstract

Two recombinant transformation vectors ZpbetaypGHcDNA (6.5 kb) and ZpbetartGHcDNA (4.9 kb) were constructed with Zp promoter at their respective 5' ends and a beta-reporter gene at their 3' ends. Freshly fertilized eggs of the Indian catfish, Heteropneustes fossilis (Bloch), were electroporated at optimum conditions (0.100 kV voltage; 100 microfarad capacitance; infinity ohms resistance and 2 pulses) in the presence of one of these transformation vectors (100 microgram circular DNA mL(-1)). Survival of the electroporated embryos averaged 56% at hatching against 70% in the control eggs. Southern and expression analyses confirmed the integration and translation of the transgene in all the developmental stages tested; a random Southern analysis confirmed the persistence of the transgene up to 12 months of age in both transformants. Stable expression of the transgene in the adult transformant (G(0)) was confirmed from 30% to 60% increase in the growth of the presumptive transgenics (compared with control) and a two- to sixfold increase in beta-galactosidase (beta-gal) activity. From ZpbetaypGH (G(0)) transformants, 5 out of 10 F(1) progeny inherited the transgene into the genome of the host. Genomic DNA extracts from tailfins of F(1) ZpbetartGH transformants were subjected to Southern analysis seven successive times after digestion with different restriction endonucleases. Consistent evidence for the genomic integration of the transgene was obtained (from 12 out of 12 F(1) positive rtGH transformants) confirming stable integration. Evidence is also given for integration at multiple but identical sites of the host genome. Expression of the transgene in terms of beta-gal activity also affirmed stable integration in F(1) progenies.