Basic and applied research on production of "double-muscle" cattle by use of a chimeric DNA/RNA oligo-nucleotide

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The following research was done for the production of transforming growth factor 8 gene (TGF8) knock-out cattle using a chimeric DNA/RNA oligo-nucleotide.1) To investigate effects of cell cycle of the recipient cells, bovine fibroblasts and cumulus cells, on the gene expression, p-β-act/luc^+/IRES/EGFP/neo^r was injected into the cell nuclei. The luminescence was highly detected when cells were in a confluent state, but not after serum starvation. To examine the efficiency of gene transformation of the cells, gene introduction methods, DNA-microinjection, etectroporation and a transfection reagent were used for introduction of the p-β-act/luc^+/IRES/EGFP/neo^r into the bovine somatic cells. The gene was transfected most effectively by use of the transfection reagent by detecting EGFP fluorescence. Furthermore, in the transfected cells, the fluorescence was further detected after selection by G418, and in nuclear transplanted-embryos with the transfected. These indicated that the gene m … More ay be integrared in genome of the cells. From the data, the use of the transfection reagent was the most effective to introduce the exogenous gene into bovine somatic cells.2) A point mutation of cyh2 encoding L29 which is a ribosomal protein in yeast induce cychloheximide tolerance. We examined whether the similar mutation of the homologous gene encoding L27, a mammalian ribosomal protein induced the cychloheximide tolerance during cell culture. We designed the chimeric DNA/RNA oligo-nucleotide which induced the mutation of L27 gene, and introduced the oligo-nucleotide into bovine and mouse fibroblasts by the transfection reagent The cells were cultured under cychloheximide for 10 days. All of the not-transfected and vecter-transfected cells were degenerated 10 days after culture with cychloxeximide, but bovine and mouse cells transfected with the oligo-nucleotide were survived even with cychloheximide for 10 days. The results showed the transfection of the oligo-nucleotide might induce the point-mutation of the L27 gene of the cells and consequently the cells acquired the cychloheximide tolerance.3) We designed the the chimeric DNA/RNA oligo-nucleotide which induced point-mutation of bovine GDF8 gene. The use of the oligo-nucleotide may provide the alternative method to produce the KO animals. Less