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Construction of Mammary Gland-Specific Vector for Sfat-1 and Its Transgenic Cells

Author: YuanTianJie
Tutor: ZhuHuaBin
School: Chinese Academy of Agricultural Sciences
Course: Animal Genetic Breeding and Reproduction
Keywords: n-3 PUFAs sfat-1 gene mammary gland bioreactor
CLC: Q78
Type: Master's thesis
Year: 2009
Downloads: 85
Quote: 2
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Abstract


The shift in n-6/n-3 fatty acid ratio, especially the deficiency of n-3 fatty acids, might have imposed a risk of modern diseases. These fatty acids are termed essential fatty acids because they could not be produced by the body (whether animal or human) and must be supplied by the diet for good health. The available sources of n-3 fatty acids is limited. Enriching the food supply with n-3 PUFA is necessary in terms of dosage and safety sources of n-3 fatty acids. The fat-1 gene encodes an n-3 fatty acid desaturase that can convert the parent n-6 fatty acid to the n-3 fatty acid.It increase the n-3 PUFA content of products endogenously. Increasing the proportion of n-3 PUFA in milk fat would help to improve the nutritional composition of an important component of people’s diet.We cloned two 5’ flanking fragment of the transcribed region of bovineβ-lactoglobulin BLG1 (1450bp) and BLG2(1575bp), The nematode gene sfat-1 which optimized the codon and pCDNA3.1(+) as the skeleton vector is used in this study. Then constructed tow mammary gland-specific vectors pCB1F1E (8976bp) and pCB2F1E(9101bp), with tow negative control vectors pCB1E (7823bp) and pCB2E (7948bp). Restriction Analysis the four vectors by Double Cut with NotⅠ. The Connections are consistent with the original design.It indicate that these four vectors are available. In order to prepare the nuclear donor cells for bovine transgenic cloning, bovine fetal fibroblast (BFFb) cells were isolated by attaching tissue explants from skin of a bovine fetal. The 8079 fetal fibroblast cell line is transferred using cationic lipid-transfer method. Transfected cells was green stimulated by blue fluorescence. The positive transgenic cells are obtained after selected in 500μg/mL and maintained in 250μg/mL G418 respectively. After testing, the PCR test proved that foreign genes has been integrated within the genome.As initial results,the transgenic cells can be good donors for somatic cell nuclear transfer. Obtained the transgenic embryo expressed fat-1 gene, lays the foundation for research of transgenic cattle. The tow negative control vectors as the universal mammary gland vectors, would help making mammary gland bioreactor with the technic somatic cell nuclear transfer.

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CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)
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