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Preparation for Recombinant Lentivirus Expressing Selected Rat SMAD3 siRNA

Author: ChenPeng
Tutor: ZhangJianJun
School: Luzhou Medical College
Course: Internal Medicine
Keywords: RNA interference SMAD3 Lentiviral vectors Liver regeneration
CLC: R575
Type: Master's thesis
Year: 2010
Downloads: 44
Quote: 0
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Abstract


Transforming growth factor-β (transforming growth factorβ TGF-β) and activin A (ACTIVIN A) are multifunctional cytokine, both signaling pathways and related functions is also recent research focus. Previous studies have shown that the TGF-β/ACTIVIN A with cell proliferation, differentiation, migration and apoptosis-related, involved in the body from the inflammatory response, tissue repair to a series of important biological processes such as embryonic development, and a closely related to the occurrence of some human diseases. Especially in the liver, the signaling pathway abnormalities with hepatitis, liver fibrosis, cirrhosis, liver cancer and liver regeneration are closely related. Therefore has important biological significance for the study of the pathway, the study of protein interactions of its components will be key to elucidate the function and regulation mechanisms. The purpose of this study is to build targets downstream protein SMAD3 TGF-β/ACTIVIN A short hairpin RNA (short hairpin RNA, shRNA) diffuse virus expression vectors provide a powerful tool to further explore the mechanism of liver regeneration. Purposes: research blocked SMAD3 signaling, and inhibition of hepatic apoptosis and promote regeneration, screened siRNA against rats SMAD3 gene fragment RNAi lentiviral vector plasmid construct targeting SMAD3 gene produces stable transcription rats SMAD3 shRNA lentiviral infection of normal rat liver cells BRL-3A. : According SMAD3 gene design and synthesis of six pairs of siRNA transfected rat liver cell line BRL-3A extract total protein using western blotting method for screening the higher one pairs of siRNA interference efficiency. Designed and synthesized according to the screening of siRNAs can be expressed shRNA ssDNA were annealed to prepare the corresponding - dsDNA ligated into after XhoI and HpaI double digestion linearized lentivirus vector plasmid PLL of 3.7 vector, identified by enzyme digestion and positive clones were sequenced plasmid extracted with lentiviral packaging plasmids PRRE, PREV, VSVG plasmid by CaCl2 generate lentivirus into human embryonic kidney cell line 293FT. Collection contains a virus supernatant infected target cells, and fluorescence microscope cell fluorescence. Results: wb results, successfully screened out a high efficiency siRNA targeting SMAD3 gene. By restriction enzyme digestion, sequencing analysis confirmed that the target sequence is successfully connected to the carrier vector was constructed successfully, packaging success. Successful infection BRL-3A cells. Conclusion; successfully constructed and screened RNAi lentiviral vector carrying rat SMAD3 gene and an effective target for experimental platform for further study of the role of SMAD3 in liver regeneration.

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CLC: > Medicine, health > Internal Medicine > Digestive and abdominal diseases > Liver and gall bladder disease
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