Transfer of growth hormone gene into fertilized egg of catfish, clarias macrocephalus, by microinjection

Abstract

Transfer of Growth Hormone gene into fertilized egg of catfish, Clarias macrocephalus Gunther., by microinjection was carried out with four objectives, 1) to compare the survival rate of microinjected egg and the control (uninjected egg), 2) to compare the growth rate between fish derived from microinjected embryos and the control, 3) to detect the introduced gene in fish blood and 4) to detect the introduced gene in various tissues of injected fish. Plasmid pXGH 5 containing the human growth hormone gene (hGH) fused to the promoter mouse metallothionein-I (mMT-I) was prepared and linearized with BamH I digestion. Microinjection of the plasmid solution about 240 pl containing 106 copies, was carried out into the germinal disc of fertilized egg of catfish at three developmental stages, i.e. one-cell stage and four-cell stage, at 25oC. The hatching rates of these eggs were 25.91 + 23.00 %, 29.71 + 29.45 % and 30.94 + 30.22 % respectively, which was not significantly different. When compared with the control group (40.67 + 20.76 %), all three stages microinjected eggs were significantly lower (P<0.05). The survival rates at one month old-fry derived from one-cell stage, two-cell stage, four-cell stage and control were 52.02 + 19.81 %, 46.67 + 30.49 %, 35.51 + 34.13 % และ 53.61 + 22.30 %, respectively, which was not significantly different. In comparing the growth rate between fish derived from microinjected egg and the control of the same spawner was performed by rearing in cages at the same densities. Only growth rate of fish, derived from one-cell microinjected egg was significantly higher than the control (P<0.05). While growth rate of fish derived from two-cell microinjected egg was higher than the control, and growth rate of fish derived from four-cell stage microinjected egg was similar to the control. Detection of the introduced gene in microinjected fish blood was carried out by using Polymerase Chain Reaction (PCR) technique, a practical method for amplifying the fragment of hGH at 186 bp in size. It was found that the integration rate of introduced gene in one-cell stage, two-cell stage, and four-cell stage microinjected fish was 5.05 % (5 of 99 samples), 6.45 % (2 of 31 samples) and 8.5 % (1 of 12 samples), respectively. Detection of the introduced gene in various of microinjected fish was performed by genomic DNA extraction and dot blot hybridization. The result found mosaicism in various tissues of fish, derived from all stages of microinjected eggs. This study found that embryonic developmental stage of yellow walking catfish at one-cell and two-cell stages were appropriate stages for gene transferring by microinjection.