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SiRNA sequence restraining bak gene expression

A gene expression and sequence technology, applied in the field of siRNA double-stranded sequence, can solve the problem of not having the most suitable siRNA sequence

Inactive Publication Date: 2008-07-16
SHANXI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve the problem that there is no optimal siRNA sequence for suppressing bak gene expression in the prior art, the present invention provides a siRNA sequence for suppressing bak gene expression

Method used

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  • SiRNA sequence restraining bak gene expression
  • SiRNA sequence restraining bak gene expression
  • SiRNA sequence restraining bak gene expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Synthesis of siRNA inhibiting bak gene expression

[0037] Using the free software available online from Ambion Corporation ( http: / / www.ambion.com / ), according to the principle of Reynolds wrote siRNA prediction program. From http: / / www.ncbi.nlm.nih.gov / Obtain the cDNA sequence of bak (GenBANK No. NM_001188) on the Internet, and then use the prediction program to predict siRNA. Based on the results of comparing the predicted siRNA sequences, some siRNAs were selected. Blast searches were carried out on both strands of these selected siRNAs (nr database of NBCI was selected as the database), and three siRNA sequences that inhibited bak gene expression were randomly selected in the search results, namely siRNA1, siRNA2, and siRNA3, and a random sequence was selected as a negative control. A, G, C, U in the bak siRNA sequence represent adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide and uracil ribonucleotide, T represents thymine de...

Embodiment 2

[0044] Example 2: In vitro transfection of bak siRNA and determination of optimal transfection dose selection and optimal action time

[0045] The transfection reagent of Ambion Company was used for transfection, and all operations were performed according to the operating procedures provided by Ambion Company. The specific steps are: the three pairs of bak siRNA sequences obtained in Example 1 and the negative control powder are respectively dissolved in RNAase-free sterile water to prepare a concentrated solution with a final concentration of 20 μmol / L, which is diluted to 2 μmol / L when used. application fluid. Neuroblastoma cells were seeded in 6-well culture plates (300,000 cells / well), cultured with 1640 culture medium containing 15% fetal bovine serum for 24 hours, and then the cell culture medium was replaced with 1640 medium containing 5% fetal bovine serum 1640 medium. For transfection, 5 μl of siRNA solution (2 μmol / L of bak-siRNA or 2 μmol / L of negative control) a...

Embodiment 3

[0052] Example 3: Detection of bak gene inhibition rate and optimal sequence screening after bak siRNA in vitro transfection

[0053] SH-SY5Y cells in the logarithmic growth phase were taken to make 1.5×10 5 Each / ml initial concentration cell suspension was added to a 24-well cell culture plate (Corning Company), and 250 μl was added to each well, and a 2 μmol / L negative control group was established, siRNA1 group, siRNA2 group, siRNA3 group, 5 wells in each group, After transfection by group, the supernatant was aspirated, trypsinized and the cells were collected. The mRNA of cells was extracted by Trizol method, the mRNA was reverse transcribed into cDNA by reverse transcription reagent, and the expression of bak gene was detected by fluorescent quantitative PCR. The specific steps are as follows: collect the cell suspension, centrifuge at 1500 rpm for 10 min, and discard the supernatant. Add 600μl Trizol, mix well, and place at room temperature for 20min. Add 0.12ml of c...

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Abstract

The invention relates to a siRNA double-strand sequence, which is a siRNA sequence for inhibiting bak gene expression. The invention solves the problem that the prior art has no siRNA sequence which is most appropriate for inhibiting the bak gene expression, the sequence is siRNA1: an antisense strand is 5'-AGUGCACACUUGCUAAAGCtt-3' and a sense strand is 5'-GCUUUAGCAAGUGUGCACUtt-3'; the siRNA double-strand sequence which is provided by the invention can be used for preparing drugs for inhibiting neural cell apoptosis or treating neurodegenerative diseases. The siRNA double-strand sequence enters into a biosome in a certain mode, which is the specific expression for inhibiting disease-related proteins and is more effective than any means; the invention can lead related disease genes to be in silence, carry out effective knockout to the expression of a target gene and achieve treatment purpose, and the specificity of the sequence and the powerful effect of the gene inhibition effect are unbeatable compared with the other drugs.

Description

technical field [0001] The invention relates to an siRNA double-strand sequence, specifically a siRNA sequence for inhibiting bak gene expression. Background technique [0002] The phenomenon of RNA interference (RNA interference, RNAi) is an evolutionarily conserved defense mechanism against the invasion of transgenes or foreign viruses, and it is a sequence-specific post-transcriptional gene silencing (PTGS). It has been found in many different species of organisms, and it is transmitted between the cells of organisms, and has the function of resisting virus invasion and maintaining genome stability. RNAi technology can not only greatly promote the development of the human post-genome project, but also screen drug target genes with high throughput, promote gene therapy, new drug development, etc., and open up new ways for the treatment of cancer, genetic diseases and other diseases. The research and application of RNAi has extremely important theoretical and practical sig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C07H21/02A61K31/713A61K48/00A61P25/28C12N15/113
Inventor 牛侨张勤丽
Owner SHANXI MEDICAL UNIV
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