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Study on Construction and Transfection of Mammary Gland Specific Expression Vector for Ssβ-gly

Author: DuXue
Tutor: ZhouHuanMin
School: Inner Mongolia Agricultural University
Course: Animal Developmental Biology and Biotechnology
Keywords: Mammary gland bioreactor Thermolysin glycosidase Breast - specific expression vector Liposomes
CLC: Q78
Type: Master's thesis
Year: 2011
Downloads: 21
Quote: 0
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Abstract


Mammary gland bioreactor production of biologically active proteins , is the hot spot of the current biological research in the field of preparation of mammary gland bioreactor key is to construct a reasonable , high efficiency expression of eukaryotic expression vector specific good , this experiment casein a major protein component of milk . For the purpose of the study , the upstream regulatory components of the bovine casein gene promoter thermolysin glucosidase gene lacS gene , build a mammary specific expression vector , and use of the reconstructed carrier liposome-mediated transfection of bovine mammary epithelial cells and Mongolia bovine fetal fibroblast cells , the identification of DNA , RNA and protein levels of the transgenic bovine mammary epithelial cells do . Of the transgenic bovine fetal fibroblast Mongolia to do DNA level identification . First, by the PCR technique from the different templates respectively amplified by the CMV promoter , the portion of the poly (A ) tail sequence , lacS gene sequence , BC promoter , connected sequentially the above sequence and the pDsRed2-1 plasmid , using the CMV promoter red fluorescent protein gene promoter directs expression to facilitate positive clones were screened ; BC promoter to guide target genes lacS specific expression in the bovine mammary ; build mammary gland-specific expression vector pBL . Secondly, the use of liposomes mediated reconstructed vector was transfected into bovine mammary epithelial cells , and were identified from the DNA, RNA and protein levels . Mongolia bovine fetal fibroblast cells cultured morphological analysis , growth curve , karyotype analysis , gender identification and G418 minimum lethal concentration determination . Somatic cells by liposome -mediated reconstruction vector was transfected into the Mongolia bovine fetal fibroblast cells , and to do DNA identification , as a follow-up nuclear transfer experiments , the production of transgenic animals . The results show that : the bovine mammary epithelial cells G418 minimum lethal concentration of 700μg/ml, able to detect the expression of the target gene in the DNA, RNA and protein levels . Mongolia bovine fetal fibroblast G418 minimum lethal concentration of 700μg/ml. The target gene can be detected at the DNA level . In summary, this expression vector construction is successful, the specific expression in the mammary gland , and source of cells for nuclear transplantation can provide for subsequent experiments .

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