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Small interference RNAs for human GSK-3beta gene and application thereof

A β gene and small molecule interference technology, applied in the field of nucleic acid, can solve the problems of low selectivity and easy side effects, and achieve the effect of low toxicity and side effects

Inactive Publication Date: 2012-11-14
SHANGHAI TENTH PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, an important problem facing the clinical application of GSK-3β inhibitors is their low selectivity and easy to cause side effects

Method used

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  • Small interference RNAs for human GSK-3beta gene and application thereof
  • Small interference RNAs for human GSK-3beta gene and application thereof
  • Small interference RNAs for human GSK-3beta gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0051] The preparation method of siRNA provided by the invention includes the design of siRNA sequence and the synthesis of siRNA sequence.

[0052] siRNA design refers to: firstly, through http: / / www.dharmacon.com / designcenter / DesignCenterPage.aspx server, select 19bp or 21bp within the gene coding region of GSK-3β gene cDNA sequence (Genbank accession number is NM_002093.2) the nucleotide sequence. The principle of selecting the 19bp or 21bp nucleotide sequence through http: / / www.dharmacon.com / designcenter / DesignCenterPage.aspx server is: (1) total siRNA double bond energy <1; (2) antisense strand 5' end binding energy 3-9; (3) sense strand 5' end binding energy 5-9; (4) GC content between 36-53%; (5) antisense strand 5' end and sense strand 5 ' The energy difference between -1 to 0. After selection according to the above principles, the candidate siRNA target sites were compared with human gene sequences by BLAST analysis to exclude those with high sequence homology with ...

Embodiment 1

[0067] Embodiment 1 siRNA design and synthesis

[0068] The human GSK-3β gene (NM_002093, NM_001146156) was extracted from the NCBI website, and the siRNA shown in Table 1 was designed using the online software http: / / www.dharmacon.com / designcenter / DesignCenterPage.aspx. The designed siRNA was entrusted to Shanghai Aibosi Biotechnology Co., Ltd. for synthesis.

[0069] Table 1 siRNA against human GSk-3β gene

[0070]

Embodiment 2

[0071] Example 2 Cell Culture and Transfection

[0072] Cell culture: human osteoblast MG-63 (purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences), cultured in 10% FBS-DMEM medium (FBS was purchased from Gibco, DMEM was purchased from Hyclone), and 2mM L-glutamine was added Amide, 37°C, 5% CO 2 cultured in a saturated humidity incubator.

[0073] siRNA transfection:

[0074] 1) Human osteoblasts MG-63 were cultured in a 10cm dish until 70-80% confluent, and seeded in a 6-well plate.

[0075] 2) Press 2.5×10 5 Inoculate a 6-well plate at the concentration of cells / well, mix well and store at 37°C in 5% CO 2 Cultivate for 24h.

[0076] 3) Take 11 sterile 1.5mL EP tubes (Group A) and add 250μl Opti-MEM I respectively, then add 100pmol siRNA (siRNA GSK-3β1-10, without siRNA as a control), and take the other 11 Add 250μl Opti-MEM I to sterile 1.5ml EP tubes (group B), respectively add 5ul of the selected transfection reagen...

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Abstract

The invention relates to a set of siRNAs directed at the human GSK-3beta gene and application thereof. According to the invention, sequences at the coding region of the human GSK-3beta gene are selected as target sites of the siRNAs, and the siRNAs are designed based on 10 to 30 (preferably, 15 to 27, and more preferably, 19 to 23) continuous base sequences in the target sites. Cell experiments prove that the siRNAs can reduce expression of the human GSK-3beta gene in a sequence-specific manner and thus reduce the expression level of GSK-3beta protein.

Description

technical field [0001] The present invention relates to the field of nucleic acid technology, in particular to RNA interference technology, more specifically to small molecule interference ribonucleic acid capable of inhibiting the expression of human GSK-3β gene and its application. Background technique [0002] RNA interference technology, also known as gene knockdown (knock-down) or gene silencing (gene silencing), is a typical post-transcriptional gene regulation method, also known as post-transcriptional gene silencing (post-transcriptional gene silencing, PTGS) . The earliest report on RNA interference appeared in 1990. Two different research groups simultaneously reported the phenomenon of RNA interference in transgenic plants, and later observed it in almost all eukaryotic organisms such as nematodes, fruit flies, zebrafish, and mice. to the phenomenon of RNA interference. In 1999, Hamilton and Baulcombe detected RNA fragments with a length of 21-25 nucleotides in ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/113A61K48/00A61P19/10A61P3/10
Inventor 盛辉曲伸于永春盛春君徐倍程晓芸李文君王吉影李鸿张戈秦岭
Owner SHANGHAI TENTH PEOPLES HOSPITAL
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