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Construction and application of GADD45betaRNA interference report vector based on EGFP

A technology of EGFP-N1 and carrier, which is applied in the construction and application of EGFP-based GADD45β RNA interference reporting carrier, can solve the problems of cumbersome and redundant experimental process, time-consuming and labor-intensive operation, and large amount of siRNA

Inactive Publication Date: 2015-09-09
SHANXI UNIV
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AI Technical Summary

Problems solved by technology

This method requires a large number of test cells, a large amount of siRNA, and RNA extraction, reverse transcription, and qPCR operations are time-consuming and laborious, making the entire experimental process cumbersome and redundant.

Method used

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  • Construction and application of GADD45betaRNA interference report vector based on EGFP
  • Construction and application of GADD45betaRNA interference report vector based on EGFP
  • Construction and application of GADD45betaRNA interference report vector based on EGFP

Examples

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Embodiment 1

[0017] Example 1: Construction of p-GADD45β-EGFP-N1 and its application in screening siRNA in bladder cancer cells

[0018] 1. Construction of recombinant reporter vector p-GADD45β-EGFP-N1

[0019] 1. Preparation of target fragment and vector backbone

[0020] Take the existing vector pcDNA3.1-GADD45β in our laboratory, use Hind III and BamHI to carry out double digestion, and use Hind III and BamHI to digest the backbone vector pEGFP-N1 at the same time, and the reaction system is carried out according to the Hind III and BamHI instructions (NEB). See Table 1.

[0021] Table 1 Double digestion system of fragments and backbone vectors

[0022]

[0023] 2. Gel recovery enzyme digestion products

[0024] The above-mentioned double enzyme digestion products were recovered by gel, and the recovery process was carried out strictly according to the instructions of the Gel Extraction Kit of OMEGA Company. Among them, the recovered fragment of pcDNA3.1-GADD45β is about 500bp, a...

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Abstract

The invention relates to construction of a GADD45betaRNA interference report vector based on EGFP and to a method of fast screening siRNA. The method can be used for studying gene function at a cellular level and an in-vivo level. By inserting a GADD45beta open reading frame into a pEGFP-N1 vector, the GADD45betaRNA interference report vector, of p-GADD45beta-EGFP-N1, based on EGFP is constructed, and fusion expression of GADD45beta and EGFP is carried out in the vector. The vector together with the synthetic GADD45betasiRNA cotransfects the bladder cancer cell line T24, and it is found out that interfering effects of GADD45betasiRNA360 and siRNA638 are best. Compared with a conventional qPCR method, the method in the invention is time saving and labor saving. The method is suitable for fast screening siRNA in bladder cancer cells and mammalian cells and is used for studying gene function.

Description

Technical field: [0001] The invention relates to the construction, construction method and application of an EGFP-based GADD45β RNA interference reporter carrier, especially for rapid screening of GADD45β-specific siRNA in bladder cancer cells and mammalian cells for gene function research. Background technique: [0002] GADD45β plays an important role in cell cycle, DNA damage repair and signal transduction. In recent years, it has been found that GADD45β is closely related to the occurrence, development and transformation of tumors, and plays an active role in tumor cell apoptosis. When studying the role of GADD45β in the occurrence and development of tumors, it is necessary to silence the GADD45β gene to study the changes in tumor cells when the gene is not expressed or the expression level is low, so as to clarify the gene function. [0003] The conventional gene silencing method is to design multiple siRNAs for the target gene, but the efficiency of siRNAs at different...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/62C12N15/113C12N5/10
Inventor 张婷婷张建珍任璐何佼马恩波郑耀武任连生
Owner SHANXI UNIV
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