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Method for knocking down fish gene expression based on heat induced miR30-ShRNA

A gene expression and fish technology, applied in the field of gene expression knockdown in fish based on heat-induced miR30-shRNA, can solve the problems of poor, irreproducible results, siRNA or shRNA knockdown research technology, etc. Effect

Inactive Publication Date: 2016-12-07
CHONGQING INST OF GREEN & INTELLIGENT TECH CHINESE ACADEMY OF SCI
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Problems solved by technology

However, recent studies have shown that the siRNA or shRNA knockdown research techniques used in zebrafish have some problems that cannot be repeated or have poor results (Kelly A, Hurlstone AF. Brief Funct Genomics.2011,10:189-196. )

Method used

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  • Method for knocking down fish gene expression based on heat induced miR30-ShRNA
  • Method for knocking down fish gene expression based on heat induced miR30-ShRNA
  • Method for knocking down fish gene expression based on heat induced miR30-ShRNA

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Embodiment

[0047] The zebrafish Apex1 gene information is retrieved from the database, and the ORF sequence of the zebrafish Apex1 gene is shown as SEQ ID NO.1.

[0048] Design of efficient siRNA targets against the Apex1 gene. The effective siRNA target sequence targeting the Apex1 gene contains the sequence shown in SEQ ID NO.2.

[0049] Specifically, the sequence shown in SEQ ID NO.2 is: GGAGAGGCTGACAATGGAA.

[0050] In this example, a sequence vector (pISceI-HSP70-miR30-Apex1-eGFP) that uses the heat shock promoter HSP70 to regulate the expression of downstream genes was constructed by using the miR30 interference sequence.

[0051] First, a single-stranded partial miR30 interference sequence and a single-stranded shApex1 were synthesized in vitro, and the target fragment miR30-Apex1 was inserted by annealing, with BbsI restriction sites at both ends of the target fragment. The insertion target fragment miR30-Apex1 contains the sequence shown in SEQ ID NO.3. The sequence shown in ...

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Abstract

The invention belongs to the technical field of biology and particularly relates to a method for knocking down a fish gene expression based on heat induced miR30-ShRNA. For solving the problem that certain results of a zebrafish siRNA or shRNA knockdown research technique for zebra fish cannot be repeated or poor in effect in the prior art, the method adopts a miR30 mediated shRNA gene knockdown system to establish a corresponding RNAi carrier and achieves the expression level control of a target gene under the heat shock induction condition by introducing a thermal induction regulation and control technique. Results show that a target sequence is an miR30 Apex1shRNA carrier shown as SEQ ID NO.1, heat shock induced knockdown of an Apex1 gene expression can be achieved, and Apex1 gene expression is remarkably reduced.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for knocking down fish gene expression based on heat-induced miR30-shRNA. Background technique [0002] The DNA base excision repair (BER) pathway mainly monitors and repairs DNA base damage induced by reactive oxygen species, alkylating agents and ionizing radiation. The BER pathway consists of multistep enzymatic reactions involving various DNA repair proteins. This pathway is mainly divided into three steps: (1) DNA N-glycosylase recognizes damaged bases, hydrolyzes the chemical bonds between N5 of abnormal purines and N3 of abnormal pyrimidines and deoxyribose, and forms on the DNA chain Apurinic / apyrimidinic site (AP); (2) AP endonuclease (APEX1) cuts a gap upstream of the AP site, making one end a 5'-phosphate group and the other end a 3'-hydroxyl group; (3 ) DNA polymerase re-synthesizes the correct base, and finally connects it by ligase. The role of A...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/89A01K67/027
CPCA01K67/0276A01K2227/40C12N15/89
Inventor 裴得胜其格乐邓顺
Owner CHONGQING INST OF GREEN & INTELLIGENT TECH CHINESE ACADEMY OF SCI
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