Integration, expression and germ-line transmission of foreign growth hormone genes in medaka (Oryzias latipes)

Abstract

Gene constructs consisting of human growth hormone (hGH) gene driven by promoter/regulatory sequence of mouse metallothionein (mMT), viral thymidine kinase (vTK), rat cholecystokinin (rCCK), or chicken beta-actin (cBA) gene were injected into the cytoplasm of fertilized medaka eggs via the micropyle. More than 49% of the injected embryos survived at hatching. Up to 26% of the survivors showed integration of the introduced gene construct, as determined by polymerase chain reaction analysis and subsequent confirmation by Southern blot hybridization of the genomic DNA. A significant fraction of F1 progeny, derived from crosses between transgenic founders and the nontransgenic individuals, inherited the transgene. Expression of hGH gene was also observed in some of the P1 founders and F1 transgenic progeny carrying mMT-hCG or cBA-hGH gene. Furthermore, the growth performance of the P1 mMT-hGH and cBA-hGH transgenic founders and F1 cBA-hGH F1 transgenic progeny was significantly greater than their full sibling, nontransgenic individuals. In addition to the microinjection experiment, a gene construct containing the long-terminal repeat (LTR) sequence of avian Rous sarcoma virus (RSV) and rainbow trout (rt) GH2 cDNA was introduced into embryos of medaka by electroporation using an exponential decay electroporator. Approximately 70% of the electroporated embryos survived at hatching, and 20% of the survived individuals integrated RSVLTR-rtGH2 cDNA into their genomes. These two techniques will greatly enhance the ability to study regulation of gene expression in transgenic animals during differentiation and development.