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Polynucleotide for target gene

A polynucleotide, target gene technology, applied in genetic engineering, plant gene improvement, application, etc., can solve the problems of difficult RNA interference effect, annealing and other problems

Inactive Publication Date: 2009-10-14
OTSUKA PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to achieve the above-mentioned purpose, it is important not only to synthesize RNA, but also to have a structure for transcribing siRNA from a DNA plasmid vector. However, it is generally considered that two different short-strand RNAs are expressed and annealed in the cell, and the target cell It is difficult to exert the effect of RNA interference

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0138] Example 1: Synthesis of polynucleotides for target genes of the present invention

[0139] In this example, the target gene uses the sequence of 19 nucleotides at positions 434-452 in the luciferase gene (Genbank Accession No. U47296) as the target site for RNA interference, and synthesizes a target gene containing two element sequences. polynucleotide.

[0140] The RNA sequence was synthesized using a commercially available automatic synthesizer (ABI3900 high-throughput DNA synthesizer manufactured by Applied Biosystem) using the phosphate amino protection method and the reagents required for synthesizing RNA. In addition, as a non-specific control group, RNAs of forward sequence (F) and reverse sequence (R) consisting of 21 bases complementary to sequence positions 936-954 of EGFP (Genbank Accession No. U55763) were synthesized.

[0141] In the thus obtained polynucleotide for target gene of the present invention, the 12-base sequence of element (II) is named uGL3.12...

Embodiment 2

[0143] Example 2: RNA function inhibitory activity experiment using polynucleotides for target genes of the present invention

[0144] 1. Preparation of RNA for RNA transfection

[0145] The uGL3.12RNA (142nM) and uGL3.7RNA (135nM) obtained above were dissolved in distilled water to prepare a 100pM / μl solution.

[0146] Then, 30 μl of the above-mentioned RNA solution, 30 μl of distilled water and 240 μl of buffer solution (100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate) were prepared into a mixed solution as the stock solution of uGL3.12RNA and uGL3.7RNA (stock solution: 10pmole / μl). The 5-fold and 10-fold dilutions were all diluted with the above buffer.

[0147] 2. Preparation of siRNA for non-specific control group

[0148] The single-stranded forward sequence (F) RNA made of 21 bases complementary to the sequence position 936-954 of the EGFP gene obtained in the above-mentioned embodiment 1 and each 10 pmol of RNA of the reverse sequence (R)...

Embodiment 3

[0166] Embodiment 3: the RNA function inhibition effect of Lamin A / C gene function inhibition carrier

[0167] In this example, the intracellular expression vector was prepared by using the 20-base sequence of the 994-1013 position of the Lamin A / C gene (Genbank Accession No. X03445) as the target site of RNA interference to evaluate its RNA function inhibitory effect .

[0168] (1) Preparation of Lamin A / C gene function inhibited cells

[0169] 1. Preparation of Lamin A / C gene function inhibition vector

[0170] The Lamin A / C gene function suppression vector was prepared as follows.

[0171] The human U6 promoter was PCR amplified using the following primers (oligos 1 and 2; Sequence Nos. 7, 8). Using this PCR amplified fragment as a template, PCR amplification was performed using oligomer 1 (SEQ ID NO: 7) and oligomer 3 (SEQ ID NO: 9), and a Csp45I restriction site was introduced to create a modified U6 promoter. The amplified fragment was double digested with restrictio...

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Abstract

The present invention provides a single-stranded polynucleotide sequence composed of the nucleic acid sequence of the complementary strand of the target gene and the element sequence.

Description

technical field [0001] The invention relates to a single-stranded polynucleotide sequence having specific RNA function inhibitory activity on target genes and a method for inhibiting target gene function using the polynucleotide sequence. Background technique [0002] The RNA interference method (RNA interference) is a method based on the phenomenon that two strands of RNA having the same sequence as a target gene (hereinafter referred to as a target gene) are introduced into cells to destroy the target gene. Transcript product (RNA), the 2 strands of RNA are composed of 2 complementary RNAs, one RNA has a sense sequence relative to the target gene, and the other RNA has an antisense sequence relative to the target gene (Fire, A. , et al., Nature, 391, 806-811 (1998)). In this specification, the case of annealing two different complementary molecules is referred to as two strands, and the case of annealing one intramolecular complementary portion or annealing two different ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/00C12N5/10C12Q1/68G01N33/50G01N33/15A61K31/7105A61K38/00A01K67/027A01H5/00C12R1/91C12N15/11C12N15/113
CPCG01N2500/10C12N2310/14C12N2310/53C12N15/111C12N15/113C12N2330/30C12N2310/111A61P1/16A61P3/10A61P9/10A61P25/00A61P25/28A61P31/00A61P31/14A61P31/18A61P31/20A61P35/00A61P35/02A61P37/06A61P43/00
Inventor 铃木干生百田裕渡边武
Owner OTSUKA PHARM CO LTD
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