Construct Human Bone Morphogenetic Protein-7 Mammary Gland Expression YAC And Integrate YAC Into Goat Somatic Cell By Spheroplast Fusion

Yeast artificial chromosom as a vector allows cloning of fragments of DNA many hundreds of kilobase in size .It can include more intacting regulatary element than traditional vectors so the expresion of the transgenic gene was controlled by the intacted regulatary elements and without being effected by the integrating position .The YAC we used containing the intacted human Lactoferrin gene ,which include the remote controlling elements and LF coding element .We modified the LF YAC through homogenous recombination .Knocking out the LF coding element and knocking into the human bone morphogenetic protein-7 full cDNA at the same time ,we obtained the hBMP7-hLF chimeric YAC .Then transfected the YAC to the goat embryonic fibroblast cell by spheroplast fusion method and obtained the cell strain which redomly intagrated the chimeric YAC .Total RNA was prepared from the human fetal liver , cDNA was synthesized as template used in PCR for amplification of the full cDNA (1.3kb) for bone morphogenetic protein-7(BMP-7) .The 5',3'flinking arms used to contract the targeted vector was amplified by PCR sequencing result showed that hBMP-7 cNDA and 3'arm 's sequence is identical to the one in the Genebank ,the 5'ann's sequence has a little difference to the Genebank ,mybe it's for the reason of individule difference .The targeted vector that we constracted is 11790bp .containing 1045bp LF 5' flinking arm ,1.3 kb hBMP-7 cDNA,720bp LF 3'flingking arm ,the selectable marker Neomycin-resistance gene and KanMX4 gene those can screened positive clone in the media including G418 ,The method using the KanMX4 as the selectable marker has a higher positive frequency about 70% than the traditional method . Then we applied the spheroplast fusion method to transfer the YAC to goat embryo fibroblast cell ,we have got a positive cell clone by 500mg/ml G418 screening ,but this clone has not transmitted to offspring cell ,so we should continue the exploration of the condition of the fusion .In this research we constracted a high effective method of modifing the YAC through homogenous recombination in the yeast .explored the method of the YAC transfected into goat fibroblast cell by spheroplast fusion and proved the possibility.of this method .