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Mouse NRDP1 gene site-specific modification system and applications thereof

A site-directed modification, mouse technology, applied in the field of genetic engineering

Inactive Publication Date: 2016-05-11
SHUGUANG HOSPITAL AFFILIATED WITH SHANGHAI UNIV OF T C M
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Overexpression of Nrdp1 can inhibit the mRNA and protein expression of lipopolysaccharide (LPS)-induced inflammatory factors IL-6 and TNFα, but can promote the production of type I interferon (IFN-β); interference with Nrdp1 can promote the expression of inflammatory cells Factor production, but inhibited IFN-β production

Method used

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  • Mouse NRDP1 gene site-specific modification system and applications thereof
  • Mouse NRDP1 gene site-specific modification system and applications thereof
  • Mouse NRDP1 gene site-specific modification system and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Target sequence design and plasmid construction of mouse NRDP1 gene TALENs

[0039] The materials and reagents required for this example FastTALETMTALENAssemblykit were provided by Sidansai Biotechnology CO., LTD (http: / / www.sidansai.com / ). The kit consists of 8 backbone carriers, 172 RVD monomer recognition modules and 5 kinds of module connection operation solutions.

[0040] In this example, for the exon1 sequence of the mouse NRDP1 gene, a recognition module was designed using TALeffectorNucleotideTarger2.0 (https: / / tale-nt.cac.cornell.edu / node / add / talen), and the T base was reserved at the 5' end. Select TALMEX-1F and TALMEX-1R as recognition modules to modify the AGCT site of mouse NRDP1 gene exon1 sequence.

[0041] TALNRD-1F: CCTCAGTAAACTTCGCCT

[0042] TALNRD-1R: TATAGCATCTTTGCTGAT

[0043] Referring to the instruction of FastTALETMTALENAssemblykit, select the corresponding connection module according to the recognition module of TALMEX-1F and TALM...

Embodiment 2

[0044] Example 2 TALENs activity detection

[0045] The amplification, extraction and purification of pTALEN-L plasmid, pTALEN-R plasmid and cell DNA were carried out according to the method of the second edition of "Molecular Cloning Experiment Guide". Resuscitate mouse 3T3-L1 cells (containing 1640 medium, 10% FBS), use lipofectacmin2000 to co-transfect pTALEN-L and pTALEN-R plasmids into mouse 3T3-L1 cells, amplify the target gene by PCR, and identify it by sequencing The mutation status of the target gene is as follows:

[0046] (1) Inoculate cells with a density of 3x105 into a 6-well plate, and prepare for transfection when the cells are confluent to 90% to 95%;

[0047] (2) Dilute 4 μg of plasmid and 10 μL of liposome in 240 μL of Opti-MEM, respectively, and place each at room temperature for 5 min;

[0048] (3) The diluted liposomes and plasmids were mixed and placed at room temperature for 20min;

[0049] (4) The mixed solution was added to the cell well containing...

Embodiment 3

[0058] Example 3 In vitro transcription to obtain the mRNA of the target gene TALENs

[0059] In the present invention, the pair of TALENs obtained in Example 2 is used as a template, mRNA is obtained by in vitro transcription, and directly injected into mouse embryos to obtain target gene-modified transgenic mice. The mouse model prepared by the present invention does not contain any exogenous screening. Gene, short cycle, low cost and high efficiency.

[0060] In vitro transcription kit mMESSAGE highyieldcapped RNA transcription kit and RNA purification kit MEGAclear TM Kits were purchased from Ambion, and the operation steps are as follows:

[0061] Preparation of template DNA: PCR template or linearized plasmid

[0062] Capped transcription reaction:

[0063] 1. Prepare the transcription system at room temperature

[0064] Dosage Element To20μL Nuclease-free Water 10μL 2×NTP / CAP 2μL 10×Reaction Buffer 1μL (optional)[α-32P]UTP a...

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Abstract

The invention belongs to the technical field of gene engineering, and provides a mouse NRDP1 gene site-specific modification system, which is the transcription activator like effector nuclease TALENs for cutting target sequence of mouse NRDP1 gene. The target sequence of mouse NRDP1 gene is represented by SEQ ID No.1. The 20 to 40th nucleotides are the recognition module TALNRD-2F. The complementary sequence of 57 to 74th nucleotide is the recognition module TALNRD-2R. The 40 to 56th nucleotides are the spacer sequence. The transcription activator like effector nuclease TALENs is composed of nuclease TALEN-L for recognizing the recognition module TALNRD-2F and nuclease TALEN-R for recognizing the recognition module TALNRD-2R; wherein the nuclease TALEN-L is protein encoded by the nucleotide sequence represented by SEQ ID No.2; and the TALEN-R is protein encoded by the nucleotide sequence represented by SEQ ID No.3. The invention also provided applications of the mouse NRDP1 gene site-specific modification system.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a mouse NRDP1 gene-directed modification system and its application. Background technique [0002] Protein degradation is mainly through two pathways: lysosomal degradation pathway and ubiquitin-mediated proteasomal degradation pathway. The lysosomal degradation pathway is a non-selective protein degradation pathway, which mainly degrades foreign proteins that enter the cell through granule uptake or pinocytosis; while the ubiquitin-mediated pathway is a specific protein that is subject to strict spatiotemporal regulation Degradation pathway, the degraded protein through E1 (ubiquitin-activating enzyme, Ubiquitin-activatingenzyme), E2 (ubiquitin-conjugating enzyme, Ubiquitin-conjungatingenzyme) and E3 (ubiquitin ligase, Ubiquitinligase) a series of actions and multiple ubiquitinligase After covalent binding, the protein is recognized and degraded by the proteasome co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/85A01K67/027
CPCC12N9/22A01K2227/105A01K2267/03C12N15/8509
Inventor 聂红明汪蓉王学斌陈建杰高月求梅昭荷申弘
Owner SHUGUANG HOSPITAL AFFILIATED WITH SHANGHAI UNIV OF T C M