| A major problem in the production of transgenic animal mammary gland bioreactors is the low production rate of high-expressing transgenic animals due to the position effect To overcome that, including all regulatory elements in the constructs of expression vector seems to be the most reasonable way. but this is very difficult when we use prokaryote-based cloning system. It have been reported that the 210 kb yeast artificial chromosome (YAC) including the human a -lactalbumin gene could direct the transgene to express in a position-independent manner, therefore is an excellent expression vector to produce transgenic animal bioreactors. But the routine methods of YAC transgene transfer such as microinjection cannot introduce YAC into livestocks by now. The rapid development of animal cloning technology provide a new way: the targeted gene was introduced into the YAC vector first, then the right YAC-bearing yeast was fused into mammalian cells, at last transgenic animals expressing high level targeted protein was produced using cloning technology.According to above consideration, experiments was carried out as below: First, targeted gene-human serum albumin(HSA) gene was obtained via PCR technology. Secondly, the HSA gene was liganded with a plasmidpRLA22 whose two arms carry DNA sequences necessary for crossing with the human a-lactalbumin YAC to form an integration vector pRLA-HSA, then the integration vector plasmid was co-transformated into the right YAC -bearing yeast with another plasmid pLRH33 which carrys a selective gene. Finally 4 right clones was obtained from 864 transformants by PCR detection. Sequencing analysis showed that the HSA gene have been introduced into the right position of the human a -lactalbumin YAC. Furthermore, works to optimize the conditions of introducing the recombinant YAC into goat f ibroblast via cell fusion was explorded . |
