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
View PDF2 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no research report on the si

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • 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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap