Method for establishing and identifying core fucosyltransferase gene silencing cell model

A gene silencing and cell model technology, applied in the field of biological function research, can solve problems such as no reports

Inactive Publication Date: 2013-04-17
DALIAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0013] At present, there is no report on the construction of Fut8 gene silencing cell model by Fut8siRNA replication-deficient recombinant retrovirus

Method used

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  • Method for establishing and identifying core fucosyltransferase gene silencing cell model
  • Method for establishing and identifying core fucosyltransferase gene silencing cell model
  • Method for establishing and identifying core fucosyltransferase gene silencing cell model

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Experimental program
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Effect test

Embodiment 1

[0044] (1) Selection of Fut8 siRNA fragments Four 21-nucleotide double-stranded siRNA sequences (19 nucleotides + overhang TT) were designed using the online http: / / rnaidesigner.invitrogen.com / sirna software program. Select 4 kinds of siRNA sequences respectively from Fut8 gene (GenBank Accession NM_016893) ( figure 1 ). Using TransIT-TKO transfection reagent (Mirus Co. Madison, USA) to transfect 4 double-stranded siRNAs into the target cells, after 24 hours, screen the most effective siRNA sequences that specifically inhibit the expression of Fut8. The ratio of TransIT-TKO transfection reagent to double-stranded siRNA is 8:3 ( figure 2 ). After measuring the enzyme activity, Fut8siRNA (759-778:CUG AUC ACU CCA GCA GAGATT) was finally determined to be the best sequence ( image 3 ).

[0045] (2) Construction of recombinant retroviral vector containing Fut8 gene The construction of pSINsi-hU6-Fut8 was designed into a double-stranded hairpin structure with siRNA hairpin Oli...

Embodiment 2

[0051] (1) Selection of Fut8 siRNA fragments Four 21-nucleotide double-stranded siRNA sequences (19 nucleotides + overhang TT) were designed using the online http: / / rnaidesigner.invitrogen.com / sirna software program. Select 4 kinds of siRNA sequences respectively from Fut8 gene (GenBankAccession NM_016893) ( figure 1 ). Using TransIT-TKO transfection reagent (Mirus Co. Madison, USA) to transfect 4 double-stranded siRNAs into the target cells, after 24 hours, screen the most effective siRNA sequences that specifically inhibit the expression of Fut8. The ratio of TransIT-TKO transfection reagent to double-stranded siRNA is 8:5 ( figure 2 ). After measuring the enzyme activity, Fut8siRNA (759-778:CUG AUC ACU CCA GCA GAGATT) was finally determined to be the best sequence ( image 3 ).

[0052] (2) Construction of recombinant retroviral vector containing Fut8 gene The construction of pSINsi-hU6-Fut8 was designed into a double-stranded hairpin structure with siRNA hairpin Olig...

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Abstract

The invention discloses a method for establishing a core fucosyltransferase gene silencing cell model, which comprises the following steps of: selecting the most effective RNA (ribonucleic acid) interference target sequence; synthesizing two sections of complementary oligonucleotides in vitro; and recombining the siRNA fragment of Fut8 gene into retrovirus plasmid pSINsi-hU6 by use of the enzyme digestion sites BamH I and Cla I on a carrier. Through 293T cell packing, the titer of the recombinant retrovirus can reach 2.1*10<5>CFU / ml, and the B cell strain is subjected to 400-1,000mu g / ml G418 screening to obtain a stable expression strain before infection of the generated recombinant retrovirus. The detection result of Real time-PCR and high performance liquid chromatography (HPLC) indicates that the activity of Fut8mRNA and enzyme is obviously reduced in the Fut8siRNA replication-defective recombinant retrovirus-infected 70Z / 3 cell. The method disclosed by the invention has the advantages of easiness in operation, high gene silencing efficiency, good repeatability and the like. The establishment of a Fut8 gene silencing cell model lays a foundation for the biological function study of Fut8 gene.

Description

technical field [0001] The invention relates to the field of biological function research, and more specifically relates to a method for establishing a core fucosyltransferase gene silencing cell model. Background technique [0002] Core fucosylation is a ubiquitous protein post-translational modification process. Fut8 is the only glycosyltransferase that modifies core fucosylation (as Figure 8 shown), can regulate the spatial structure and biological functions of proteins, such as intermolecular interactions, mutual recognition between cells, correct positioning of proteins, cell signal transduction, etc. Fut8 - / - Mice have an emphysema phenotype, which is because the core fucosyl defect on the transforming growth factor 1 receptor reduces the binding ability of TGF 1 to the receptor, activates the expression of matrix metalloproteinase (MMP), and causes lung gas Tumor lesions; Fut8 gene knockout led to a decrease in the expression of vascular endothelial growth factor r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10C12Q1/68C12Q1/48
Inventor 李文哲金锦花
Owner DALIAN UNIV
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