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Construction and application of RNA interference expression vector of targeted ZNF268 gene

A technology for expressing vectors and genes, applied in the field of molecular biology, can solve the problems of unsuitability for large-scale preparation, time-consuming, difficult transfection of PCR products, etc., and achieve the effect of reliable and convenient research means

Inactive Publication Date: 2011-12-28
WUHAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unlike siRNA expression vectors, SECs do not require time-consuming steps such as vector cloning and sequencing, and can be directly obtained by PCR. However, the main disadvantage is that PCR products are difficult to transfect into cells and are not suitable for large-scale production.

Method used

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  • Construction and application of RNA interference expression vector of targeted ZNF268 gene
  • Construction and application of RNA interference expression vector of targeted ZNF268 gene
  • Construction and application of RNA interference expression vector of targeted ZNF268 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1: Construction of the RNA interference plasmid vector for the ZNF268 gene

[0030] 1. Design and synthesis of oligonucleotide sequences expressing ZNF268 gene siRNA

[0031] The ZNF268 gene mRNA has 7 exon regions. According to its nucleotide sequence, several siRNA sequences targeting the ZNF268 gene mRNA sequence are designed and screened, and they are separated to form an auxiliary sequence (loop region) connection of a "hairpin" structure. Form the sense strand of the "hairpin" siRNA, and add linker sequences targeting pSilencer 2.1-U6 neo at both ends, the complementary sequence of the sense strand acts as the antisense strand, and add targeting sequences at both ends of the antisense strand The linker sequence of pSilencer 2.1-U6 neo anneals to form shRNA with linker for pSilencer 2.1-U6 neo.

[0032] Three pairs of siRNA sequences were obtained through further screening. Since they are respectively located on exons No. 3, No. 5 and No. 7 of the ZNF...

Embodiment 2

[0054] Example 2: Detection of interference efficiency against ZNF268 gene mRNA interference plasmid

[0055] 1. HeLa cell culture, plasmid transfection and extraction of total RNA and total protein

[0056] 1. The HeLa cell line comes from the China Center for Type Culture Collection (CCTCC, Wuhan), and is subcultured and preserved by our laboratory according to conventional methods.

[0057] 2. Using Sofast cationic polymer transfection reagent (purchased from Xiamen Sunma Company, China), three kinds of interference plasmid vectors that interfere with ZNF268 gene expression were transferred into HeLa cells. The specific transfection steps refer to the actual instructions, which are briefly described as follows:

[0058] 1) Cell seeding: In order to obtain the best transfection efficiency, the cell density should be 50-80%. It can vary with different cell lines, and the cell density is generally 60-70% for the most commonly used cell lines. For 24-well plates, the ideal ...

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Abstract

The invention belongs to the field of molecular biology and discloses a construction method of an RNA interference expression vector of a targeted ZNF268 gene and application thereof. In the method, screened siRNA oligonucleotide segments of the targeted ZNF268 gene are inserted into a vector system to construct an RNA interference plasmid vector of the targeted ZNF268 gene. Proved by experiments, the constructed expression vector can efficiently express a specific siRNA sequence in a tumor cell, and the sequence can be combined with an amRNA of the ZNF268 gene to ensure that the expression quantity of the ZNF269 gene is obviously reduced so as to achieve the purpose of suppressing the expression of the ZNF268 gene.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to the construction and application of small interfering RNA for ZNF268 gene and its expression vector. Background technique [0002] The ZNF268 gene is a typical KRAB / C2H2 zinc finger protein gene discovered in 2001. Many related research results show that: 1) ZNF268 gene plays a key role in the differentiation and development of organisms, and even in the occurrence and development of many diseases (including leukemia, tumor, inflammation, etc.); 2) because ZNF268 gene Alternative splicing exists, and its splicing isoforms have a specific expression in the blood cells of leukemia patients. Therefore, the ZNF268 gene splicing isoforms can be used in the preparation of drugs for the treatment or prevention of leukemia, and can be quickly and accurately judged Whether there is leukemia (CN101422617A; Zhao ZZ, et al. Oncol. Rep. 2008, 20, 1243-8). [0003] In order to better analyze t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/63C12N15/66A61K48/00A61P35/00A61P35/02A61P29/00
Inventor 李文鑫郭明雄汪维赵舟宙曾艳
Owner WUHAN UNIV
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