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Construction of pcDNA3.1（一）/hTGF β3 and the Stable Expression System of NIH3T3 Fibroblast with hTGF β3 and the Study on the Proliferation of the Stable Transfection of NIH3T3 Fibroblasts

Author: XieSiTian
Tutor: TangShiJie
School: Shantou University
Course: Burn Plastic Surgery
Keywords: TGFβ3 Gene transfection Gene recombination Fibroblasts
CLC: R346
Type: Master's thesis
Year: 2005
Downloads: 107
Quote: 0
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Abstract


Objective 1.To construct eukaryotic expression vector of human transforming growth factor β3（pcDNA3.1 （ - ） /TGF β3）. 2.To construct and identify the stable expression system of NIH3T3 fibroblast with hTGF P 3. 3.To verify hTGF β3 expression in the stable expression system of NIH3T3 fibroblasts and investigate its proliferation.Methods A eukaryotic expression vector for hTGF P 3 （pcDNA3.1 （—） /TGF β3） was constructed by use of recombinant DNA technique. The stable transfection of NIH3T3 fibroblasts with recombinant plasmid expressing hTGF P 3 was established by using LipofectamineTM2000 and G418 selection. The mRNA expression of TGF β 3 was detected by the RT-PCR method. MTT were adopted to examine the biology of the stable transfection of NIH3T3 fibroblasts.Results Digestion of the recombinant plasmid with BamHI and HindIII liberated DNA fragments with expected size .The sequence of DNA fragment is identifical to that published in GenBank. After G418 selection and RT-PCR analysis, 7 out of 10 cell lines transfected with pcDNA3.1 （—） /TGF β3 expressed with very high level of TGF β3,as compared with vector control transfectants that showed no expression, and compared with the other cell lines that expressed relatively low level. The stable transfection of NIH3T3 fibroblasts growth slowed down, especially on 4th to 5th days . Conclusion The eukaryotic expression vector of human transforming growth factor β3 （pcDNA3.1 （-） /TGF β3） and the stable expression system of NIH3T3 fibroblast with hTGF β3 were constructed successfully . The transgene of TGF β3 could inhibit the proliferation of NIH3T3 fibroblasts in vitro.

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