Dissertation > Excellent graduate degree dissertation topics show

The Creation of a Tissue Specific Expression Connexin 31 Construct

Author: ZhuHuaiXu
Tutor: ZuoLingQian；PanQian；LongZhiGao
School: Central South University
Course: Genetics
Keywords: Of gap junctions (gap junctions) Gap junction proteins ( Connexin ) Construction of mutants Connexin31 Keratin14
CLC: Q78
Type: Master's thesis
Year: 2008
Downloads: 59
Quote: 0
Read: Download Dissertation

Abstract

Background: The gap junction protein (Connexins) are a class of gap junction channels (Gap Junctions) mediated by small molecules between adjacent cells (<1000Da) family of proteins in the cell membrane assembly. Of these substances and the exchange of information, making the cell a coordinated response to external stimuli. Connexin31 (CX31) is a gap junction protein family, whose mutation can lead to significant, the recessive red skin keratosis (dominant and / recessive EKV), significantly recessive sensorineural deafness (dominantand / recessive hearing impairment), or the dominant nerve deafness - peripheral neuropathy (dominanthearing impairment combined with peripheral neuropathy), etc.. CX31 gene knockout mice found no deafness or skin phenotype transgenic mice study found people CX31 protein promoter F137L mutation of mouse epidermal partial horny. CX31 maintain cell homeostasis, cell proliferation, differentiation plays an important role. But CX31 channel assembly, channel permeability channel function regulation and the intracellular transport CX31 molecular characteristics and the understanding of cell behavior is still very rough. Objective: To enhance CX31 expression levels in the cell, particularly the expression level of the mutant in a particular tissue, will make it possible to the appropriate animal model phenotype. Used in this study Keratin14 protein promoter constructs CX31 wild type and mutant expression vector F137L and study of the carrier of the skin tissue expression and specific expression. To obtain an expected the CX31 vector for tissue specific expression of the skin of the transgenic animal model. Methods: We used the PCR technique, connected to CX31 product containing Keratin14 promoter, rabbit globin and Keratin14 the 3 'non-translated region of the vector. Then the vector the Lipifectamine LTX transfection Hacat cells, application immunoprecipitation and immunofluorescence technique to detect the expression of CX31. In order to study its tissue specificity, we also used the calcium phosphate method transfected HEK 293T cells and CX31 expression detected by immunofluorescence. Results: confirmed by restriction analysis and sequencing we get to the CX31 wild-type and F137L mutants carrier; CX31 expression detected by immunoprecipitation and immunofluorescence Hacat cells, and wild-type CX31 form gap junction plaques (gap junction plaques,), F137L The mutant proteins can not; immunofluorescence detection of HEK293T cells are not found to Keratin14 promoter carrier CX31 expression, while transfection pCDNA3.1A is possible by (-)-CX31-myc HEK 293T cells the positive control immunofluorescence detection of protein expression and the formation of gap junction plaques. Conclusion: In this study built CX31 tissue-specific expression vector can effectively express linearizing removal of part of its skeleton could be used for the construction of transgenic animal models.

Related Dissertations

CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)
© 2012 www.DissertationTopic.Net Mobile