| With the development of transgenic animal technology, the research of chicken oviduct bioreactor has been one of the foci of transgenic study. In this research, with the chicken ovalbum in 5 '-flanking regulatory region being successfully cloned, the plasmid expression vector pOV-B of human erythropoietin(hEPO) was constructed and was embedded in liposome. Then the donor cells were manipulated genetically and were microinjected into the recipient blastoderm at the same developmental stage. The Surrogate eggshell culture was used. Transgenic chicken embryos integrated exogenous gene were gained . It laid the foundation for making chicken ovalbumin bioreactor of hEPO.At first, the genome of chicken oviduct tissue was extracted .The specific primer was used to amplify ovalbumin 5 ' -flanking regulatory region. After gel electrophoresis , it was primarily identified as the target gene, it was cloned into pMD-18T vector .To further identify whether ovalbumin 5 '-flanking regulatory region can control exogenous gene or not, it was sequenced. The result showed that the major cap site , TATAbox , upstream promoter element (UPE), TATAbox-1ike sequence, imperfected plain dromes and steroid-dependent response element hadn ' t changed .So as a promoter, the regulatory sequence can be used to promote exogenous gene expression in thechicken oviduct. On the base of identification of plasmid pBCE, pOV-B was constructed.The cattle a -si~tyrosine promoter was cut out from pBCE by Sail and Hindlll,then the expression vector was constructed by linking ovalbumin 5 ' -flankingregulatory region with the remained plasraid pBCE without the cattle a-Si-tyrosine promoter. The detection of constructed plasmid vector which wasdigested with restriction endonuclease indicated that ovalbumin 5-flanking regulatory region had been linked with hEPO upstream and two geneshad been linked at same direction . It showed that the plasmid expressionvector pOV-B of hEPO was constructed successfully.The plasmid pOV-B was embedded in liposome. The liposome-pOV-B complex and the blastoderm cells of donor embryos were co - cultured in vitro for 0. 5h under 37'C, 5% CO:. Then they were microinjected into the recipient blastoderm of the same developing stage. 450 recipient embryos were cultured with surrogate eggshell. 22 chickens were hatched out and two chickens had melanin in head and two legs .While hatching, died embryos were selected. Embryos died before the fourth day were preserved in whole. When the embryoes died between the fourth day and the seventh day, they were dissected and their two sexual glands were preserved because they had ' t divided sexually. If embryos died between 8th days and the 21th days, their left sexual glands were preserved because the left one was bigger than the right one. The live chickens were also dissected and their eyes, muscle, brain, lung, intestine, stomach, liver, left sexual gland, feather follicle and blood were preserved so as to be detected.A pair of specific primer of hEPO gene were designed to detected all samples preserved . Of 564 samples, there were 89 positives detected from 213 samples before fourth day, and the positive rate was 41.78%; there were 4 positives detected from 73 samples between the 4th day and 7th day , positive rate was 5. 48%; there were 2 positives detected from 48 samples between the 8th day and the 21th day , positive rate was 2. 25%. None positive in all live chickens. The positives gathered in died embryos before sixteenth day.In order to further identify whether exogenous gene had integrated into chicken genome, dot blot hybridization was made in the positives between 4th day and 16th day. There were 4 positives. These positives gathered in died embryos before the tenth day.It can be seen from the above result that hEPO gene had already integrated into reproductive system. But whether it integrated into sexual line cell system or not wasn ' t known. Although the study hasn ' t gained transgenic live chicken ,it obtained early transgenic emb...
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