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Research on Transfection of Mouse Fibroblasts by UHS-spider Dragline Silk Protein Dimmer

Author: LiuXin
Tutor: AnTieZuo
School: Northeast Forestry University
Course: Developmental Biology
Keywords: Mouse fibroblasts Ultra - high sulfur keratin promoter Spider - protein gene Gene transfection
CLC: Q78
Type: Master's thesis
Year: 2009
Downloads: 36
Quote: 2
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Abstract


In order to speed up the establishment of the use of transgenic technology through new methods of animal coat for spider silk protein , this study mice model established by the conventional method based on mouse fibroblast cell line -specific promoter , followed by the skin of mice cloning, connected to the spider silk protein gene expression vector transfected into mouse fibroblasts, procedures , and explore the possibility of a transfer the spiders protein gene dimer positive cells . The results are : ( 1 ) using conventional methods , the use of fresh or refrigerated 96h within the fetal rat skin mouse fibroblast cell lines established after primary and subculture . (2) silk protein gene monomer synthetic spider dragline silk protein gene sequence in GenBank , the use of different restriction sites dimerization , BglII, NcoI and BamHI, NcoI , respectively double digested constructed spider dragline silk protein gene dimer plasmid ; ( 3) successfully cloned specifically expressed in mouse skin ultra high sulfur keratin (UHS) promoter . Connected with pAcGFP1-N1 vector and pβgal-Basic vector , the UHS-GFP and the UHS-β-gal expression vectors were constructed ; the UHS-GFP constructed having the activity of cultured mouse fibroblasts , while UHS- β-gal expression vector expression in fetal rat skin block organization activity . (4) less than 5000bp and 1000 ~ 2000bp strip obtained by BglII and BamHI double digested PCR identified about 700bp the UHS strip of the UHS-2S-3.1 expression vectors used to verify successfully constructed . (5) fibroblasts after transfection , G418-resistant the filtered amplified positive cells, and their genomic DNA by PCR to confirm the genomic integration target gene ( spider silk protein gene dimer) . The above results for the further development the positive cells genetically spider silk protein gene screening and the establishment of cell lines , transgenic mice obtained through somatic cell nuclear transfer technology and lay the foundation to establish a new method of animal coat spider silk .

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