Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

RNA interference sequence inhibiting mouse TLR9 expression and application thereof

A mouse and sequence technology, applied in the field of molecular biology, can solve the problems that cannot reflect the actual situation of the TLR9 pathway, and it is difficult to obtain TLR9

Inactive Publication Date: 2010-09-01
NANJING UNIV
View PDF0 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the function of dendritic cells in TLR9-deficient mice may be affected by the loss of TLR9-expressing cells in the microenvironment during development, and the study of CpG-TLR9 by removing MyD88 cannot reflect the actual situation of the TLR9 pathway. Access to TLR9-null mice or other materials to study the TLR9 pathway

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
  • RNA interference sequence inhibiting mouse TLR9 expression and application thereof
  • RNA interference sequence inhibiting mouse TLR9 expression and application thereof
  • RNA interference sequence inhibiting mouse TLR9 expression and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Embodiment 1: the design of siRNA sequence

[0016] According to the online synthesis software of Invitrogen, according to the mouse TLR9 [Gene Bank number: NM_031178] sequence, after selecting the open reading frame, the GC content is 40% to 60%, and the Blast search excludes the sequence homologous to other sequences as the target siRNA fragment. The sequence is: 5'-UGUAAGAACUUCAAGUUCA-3'.

Embodiment 2

[0017] Embodiment 2: the construction of interference carrier

[0018] Construct two Oligo single strands corresponding to the siRNA9.1 sequence (such as figure 1 ), each of the two chains contains a Loop sequence, and the two ends contain BamHI and HindIII restriction sites respectively, synthesized by Invitrogen Company (Shanghai). The double-stranded complex obtained by the annealing of the synthesized two strands was ligated into the vector pSilencerTM4.1-CMV neo with T4 DNA ligase (the plasmid map is as follows: figure 2 , containing G418 resistance), the clone was transformed into competent Ecoli, a single clone colony was picked, and the bacteria were shaken overnight. The extracted plasmid was subjected to HindⅢ and BamHI double enzyme digestion, and the digested product was subjected to polyacrylamide gel electrophoresis. The electrophoresis results showed that the position of the band was correct (such as image 3 ). At the same time, the plasmid was sequenced an...

Embodiment 3

[0019] Example 3: Cell culture and transfection

[0020] Raw264.7 cells (mouse macrophage cell line) were inoculated in DMEM complete medium (containing 10%, 100 U / ml penicillin, 0.1 mg / ml streptomycin), and cultivated at 37 ° C in a 5% CO2 incubator. The cells were subcultured every 2-3 days, and the cells in the logarithmic growth phase were used in the experiment. The experiment was divided into 3 groups: control group, pS4.1 group, and pS4.1-siRNA9.1 group. According to the operating instructions of Lipofectamine 2000, the ratio of Lipofectamine 2000 to plasmid was 1 μg: 2 μl. After 4 hours, the cell culture medium was replaced with DMEM complete medium. After 48 hours, G418 (final concentration: 400ng / ml) was added to the medium for selection. Cells that failed to be transfected with the plasmid will gradually die, and the concentration of G418 was maintained until the selection. Stable growing cells were obtained, that is, positive cell lines containing plasmids were o...

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 belongs to the field of molecular biology, and discloses an siRNA segment capable of inhibiting mouse TLR9 expression and a vector built on the basis of the segment. A sequence of the siRNA segment is 5'-UGUAAGAACUUCAAGUUCA-3'; two Oligo single strands of which two corresponding ends have BamHI and Hind III restriction enzyme cutting sites are synthesized according to the sequence, and are annealed continuously and connected to the vector to build an interference vector pSilencerTM4.1-siRNA9.1. Specific examples, such as RT-PCR, western blot and the like, validate that the interference vector can be used for inhibiting the expression of a mouse Raw264.7 cell TLR9 and block a signal channel caused by the TLR9 successfully and specifically. Therefore, the siRNA9.1 sequence and the interference vector obtained by the siRNA9.1 sequence can be used for obtaining a large number of mouse cells with low TLR9 expression cells and blocking the TLR9 signal channel economically, fast and conveniently.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to an siRNA interference sequence capable of inhibiting the expression of mouse TLR9 and its application. Background technique [0002] Toll-like receptor 9 (TLR9) is an important member of the Toll-like receptor family, as an important pattern recognition receptor (PRR) in innate immunity, it can specifically recognize unmethylated CpG in bacterial or viral DNA Motif, mediates clearance of viruses and bacteria. After TLR9 recognizes CpG, it activates the NF-κB signaling pathway, initiates the transcription of downstream genes, and causes a Th1-like inflammatory response, including inducing the functional maturation of B cells and DCs, and stimulating various immune cells to produce cytokines and chemokines. TLR9 not only has important biological significance in the process of mediating exogenous CpG to activate innate immunity, but also participates in the recognition of "self" and ...

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): C12N15/11C12N15/85
Inventor 侯亚义马玲李晓曦
Owner NANJING UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com