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mDHCR24 interference target spot sequence and application thereof

A technology of target sequence and RNA interference, which is applied in the field of molecular biology and biomedicine, can solve the problems of no uniform determination of the most effective target and great differences, and achieve the effect of inhibiting cytotoxicity and toxicity

Inactive Publication Date: 2011-01-12
LIAONING UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The inhibitory effects of different targets vary greatly, and there is no unified principle for determining the most effective target.

Method used

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  • mDHCR24 interference target spot sequence and application thereof
  • mDHCR24 interference target spot sequence and application thereof
  • mDHCR24 interference target spot sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The acquisition of embodiment one siRNA

[0028] by Figure 1A The four mRNA sequences underlined in the shown mDHCR24 sequence, namely: 5-GCACAGGCATCGAGTCATC-3, 5-GCACGGGTTCCAAATGTTA-3, 5-GCGCCTGGGTGGTGTTCAA-3, and 5-ACTCAGACCTGTTCTATGC-3 are the RNA interference target sequences of mDHCR24, corresponding to siRNAs were designed for each RNA interference target sequence and obtained by chemical synthesis from Dharmacon to obtain four siRNAs.

Embodiment 2

[0029] Example 2 Combined application of siRNAs complex and liposome transfection reagent DharmaFECT 1

[0030] In this example, the liposome transfection reagent used DharmaFECT 1, a special reagent for RNA interference from Dhamacon, and siGENOME Control siRNA from Dharmacon was used as a control for RNA interference. The effectiveness of RNAi interference was verified by semi-quantitative PCR, and its cytotoxicity The detection adopts differential microscope observation and cell counting method to evaluate.

[0031] Using the four siRNAs obtained in Example 1, the four siRNAs were combined to form a siRNAs complex and used in combination with liposome transfection reagent DharmaFECT 1.

[0032] (1) The toxicity of liposome transfection reagent DharmaFECT 1 to cells.

[0033] N2A cells were trypsinized according to 2.5x10 4 / cm 2 The density was planted in a 12-well plate, cultured in medium without antibiotics, and liposome transfection was performed 24 hours later. The...

Embodiment 3

[0045] Example 3 Combined application of siRNAs complex and liposome transfection reagent siPORT Lipid

[0046] Using the four siRNAs obtained in Example 1, the four siRNAs were combined to form a siRNAs complex and used in combination with the liposome transfection reagent siPORT Lipid.

[0047] In this example, in order to prove the versatility of siDHCR4 in reducing the cytotoxicity of liposome transfection reagents, the siPORT Lipid produced by Ambion was used as the liposome transfection reagent, and the siRNA targeting Ambio's cell caveolin1 was used (ID53643) was used to test the target siRNA, and was transfected according to the transfection conditions of Ambio Company. The effectiveness of RNAi interference was verified by semi-quantitative PCR method, and its cytotoxicity was detected by differential microscope observation and cell counting method.

[0048] N2A cells were trypsinized according to 2.5x10 4 / cm 2 The density was planted in a 6-well plate, cultured w...

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Abstract

The invention relates to an mDHCR24 interference target spot sequence and application thereof. The invention provides four effective RNA (Ribonucleic Acid) interference target spot sequences for the mRNA sequence of mDHCR24 to design and synthetize four siRNAs, the four siRNAs and a liposome transfection reagent are applied together in the research on the horizontal liposome transfection of the cellular level so that the cytotoxicity generated in the liposome transfection is effectively inhibited when the liposome transfection efficiency and RNA interference efficiency are ensured, thereby laying the foundation for the development of the commercial application of the target reagent as a reagent for inhibiting the cytotoxicity of the liposome transfection reagent.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and biomedicine. Four targets for RNA interference on mRNA involving mDHCR24, and siRNAs for these targets obtained in various ways. One or more combinations of these four siRNAs are used in combination with liposome transfection reagents in the preparation of attenuated liposome transfection reagents to reduce liposome toxicity. Background technique [0002] DHCR24 (24-dehydrocholesterol reductase, 24-dehydrocholesterol reductase) is an enzyme in the last step in cholesterol biosynthesis, which catalyzes the conversion of cholesterol to cholesterol. There are two sources of cholesterol in the human body, namely exogenous cholesterol from food and endogenous cholesterol synthesized by the liver and small intestine under the promotion of related enzymes. Among them, exogenous cholesterol accounts for 40%, while endogenous cholesterol accounts for 60%. Under precise regulation, the conc...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/113C12N15/88
Inventor 芦秀丽高兵曹向宇刘剑利刘振青侯芳芳王旭德
Owner LIAONING UNIVERSITY
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