Abstract
To assess the effects of different transgene forms on integration efficiency, plasmids containing human growth hormone gene under the control of the metallothionein promoter (pMThGH) in circular, linearized forms and lyposome trapped linearized plasmid were introduced into fertilized loach eggs. Prior to microinjection, plasmids were identified for the presence of human growth hormone (hGH) gene by digestion with restriction enzymes (EcoRI, BamHI, BglII) and by polymerase chain reaction (PCR). The linearized form of pMThGH was generated by digesting circular plasmids with BamHI. Liposome trapped linearized plasmid was prepared in the presence of dioleyl phosphatidyl choline, cholesterol and pMThGH. The presence of hGH genes in the genomic DNA of loach developed from microinjected eggs was identified by a PCR method using two hGH-specific primers. PCR products of the positive individuals showed one band corresponding to 479 bp, while those of negative and control individuals showed no corresponding band. Integration rates of hGH genes into fish chromosomes were 6%, 12% and 11% for circular, linearized plasmids and lyposome trapped linearized plasmid, respectively. In accordance with above order, the average body weight of transgenic loach at 6 months was increased by 1.63, 1.73 and 1.68 times over controls, respectively. The results indicate that plasmids in linearized form and lyposome trapped linearized plasmid were more efficiently integrated into loach chromosomes than circular ones, but there was little significant difference in body weight among the 3 groups of transgenic fish.
