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SiRNA interfering GDF9 gene expression and application thereof

A technology for gene expression and expression vectors, applied in the fields of molecular biology and bioengineering, to achieve good silencing effects and reduced protein expression

Inactive Publication Date: 2013-03-13
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no research report on the siRNA of buffalo growth differentiation factor 9

Method used

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  • SiRNA interfering GDF9 gene expression and application thereof
  • SiRNA interfering GDF9 gene expression and application thereof
  • SiRNA interfering GDF9 gene expression and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Construction of pCDNA3.1-GDF9 eukaryotic expression vector

[0038] 1. Synthesize the buffalo growth and differentiation factor 9 (GDF9) sequence (GenBank accession number NM_174681), connect it to the vector pUC19, and cut it into Eco RI and BamH. double digestion ( Eco R I and BamH) and sequencing proved that the synthesized fragments were correct (see figure 2 ).

[0039] 2. Construction of pCDNA3.1-GDF9 eukaryotic expression plasmid

[0040] (1) Enzyme digestion reaction

[0041] reaction system: Eco R I and BamHI double-digest the PCR product. Eco R I and BamHI double-digest pcDNA3.1(-), enzyme digestion system: 10×Buffer K 2 μL, Eco R I 1 μL, BamHI 1 μL, plasmid 6 μL, distilled water 10 μL, total volume 20 μL. Reaction conditions: act at 37°C for 1.2h, and perform 1% agarose gel electrophoresis. The digested products were purified with a DNA purification kit, and finally the DNA was eluted with 30 μL of water and stored at -20°C.

[004...

Embodiment 2

[0046] Example 2 Construction of pshRNA-copGFP Lentivector lentiviral interference plasmid

[0047] 1. According to the method and principle of buffalo growth and differentiation factor 9 mRNA sequence design, target sequences were screened out, and siRNA was designed to reversely complement buffalo growth and differentiation factor 9 gene mRNA, which were named GDF9-1, GDF9-2, and GDF9-3, respectively. Its sense strand sequence is as follows:

[0048] GDF9-1: SEQ ID NO: 1;

[0049] GDF9-2: SEQ ID NO: 2;

[0050] GDF9-3: SEQ ID NO:3.

[0051] 2. According to the siRNA target sequence, design the DNA sequence required to construct the siRNA vector that inhibits the expression of buffalo growth differentiation factor 9 gene: GDF9 shRNA template, introduced at both ends Bam H I and Eco The R I enzyme cutting site was sent to Shanghai Bioengineering Technology Service Co., Ltd. for synthesis. The specific sequence is as follows (F indicates the sense strand, R indicates th...

Embodiment 3

[0064] Example 3 Screening of Effective Targets of RNA Interference

[0065] 1. Transfection of eukaryotic cells with three lentiviral interference plasmids

[0066] pcDNA3.1-GDF9 and LV-siRNAⅠ, LV-siRNAⅡ and LV-siRNAⅢ were co-transfected into CHO cells for expression. CHO cells were co-transfected with pcDNA3.1-GDF9 and LV-shRNA-GFP empty plasmids as a control, in order to be consistent with the transfection background of the treatment group, and 6 replicates were set for each treatment. The ratio of pcDNA3.1-GDF9 to lentiviral interference plasmid is 1:1.

[0067] Lipofection was carried out according to the instruction manual of Polyfect (purchased from QIAGEN).

[0068] (1) One day before transfection, inoculate cells in a new six-well culture dish, and completely blow off the cells to ensure that the cells are evenly distributed. When transfecting, the cells should ideally cover 40-90% of the dish.

[0069] (2) Add pcDNA3.1-GDF9 and lentivirus interference plasmid a...

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Abstract

The invention discloses an SiRNA inhibiting the gene expression of a buffalo growth differentiation factor GDF9, and belongs to the technical field of gene engineering. The positive-sense strand of the siRNA has any sequence indicated by SEQ ID NO: 1-3. The invention also discloses an shRNA encoding gene containing the siRNA and slow virus interference plasmid pshRNA-copGFPLentivector constructed by the same. Through real-time quantitative Real-time PCR (Polymerase Chain Reaction) detection, a relative expression quantity is calculated according to a formula, wherein the buffalo GDF9 mRNA inhibiting efficiencies of two siRNA segments respectively exceed 100%, and the CDF9 protein expression quantity detected by the ELISA (enzyme-linked immuno sorbent assay) is also correspondingly reduced. The siRNA segments and the expression vector thereof can be used to prepare a preparation inhibiting the expression of buffalo GDF9, and can significantly reduce the GDF9 gene expression quantity.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and bioengineering, in particular to an siRNA fragment for inhibiting the expression of buffalo growth and differentiation factor 9 (GDF9) gene. Background technique [0002] RNAi (RNA interference), that is, RNA interference, is a ubiquitous monitoring mechanism in eukaryotes to resist virus invasion, inhibit transposon activity, and regulate gene expression. RNAi is a phenomenon or mechanism in which double-stranded RNA induces the degradation of its homologous mRNA, resulting in post-transcriptional silencing of the target gene. This phenomenon was first discovered in 1998 in a study of nematodes. siRNA is the exogenous dsRNA or endogenously produced dsRNA that enters the cell, and is cleaved by Dicer enzyme into small molecules with a length of 2l-23nt. This siRNA is a key intermediate product in the RNAi mechanism and has a very strong RNAi effect. [0003] In the RNAi pathway, t...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/63C12N15/867A61K48/00A61P43/00
Inventor 习欠云张永亮施振旦肖敏蕉莉石德顺刘庆友
Owner SOUTH CHINA AGRI UNIV
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