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

SgRNA (Small Guide Ribonucleic Acid) targeting sequence of specific target pig IRS1 (Insulin Receptor Substrate 1) gene and application thereof

A kind of specific and genetic technology, applied in sgRNA guide sequence and application field, can solve problems such as embryonic developmental delay, achieve the effect of eliminating expression and high safety

Inactive Publication Date: 2017-10-03
NORTHEAST AGRICULTURAL UNIVERSITY
View PDF1 Cites 32 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

IRS-1 gene is an important substrate of insulin receptor and insulin-like growth factor 1 (IGF1) receptor, in order to verify the physiological role of IRS-1 gene in vivo, gene targeting produced IRS-1- / - genotype mice, IRS-1- / - mice can be born in a normal proportion, but their embryonic development and postnatal development are obviously delayed, even if they show some insulin resistance, the overall blood sugar level can be maintained normally

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
  • SgRNA (Small Guide Ribonucleic Acid) targeting sequence of specific target pig IRS1 (Insulin Receptor Substrate 1) gene and application thereof
  • SgRNA (Small Guide Ribonucleic Acid) targeting sequence of specific target pig IRS1 (Insulin Receptor Substrate 1) gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] (1) sgRNA design

[0028] According to the genome sequence of porcine IRS1 gene (gene ID: NM001244489), a sgRNA targeting porcine IRS1 gene was designed. The 20nt oligonucleotide sgRNA guide sequence is: IRS1-sgRNA1: 5'- CGTAGTACTCGAGGCGCGCG-3', located in the exon coding region of the gene IRS1; add CACC to its 5' end to obtain the forward oligonucleotide sequence; Obtain its corresponding DNA complementary strand according to the guide sequence, and add AAAC to its 5' end to obtain a reverse oligonucleotide. Synthesize the above-mentioned forward oligonucleotides and reverse oligonucleotides respectively, and synthesize the forward and reverse sgRNA oligonucleotide sequences: denature at 95°C for 5 minutes, and anneal at 72°C for 10 minutes; after annealing, sticky ends can be formed The specific oligonucleotide sequence is shown in Table 1.

[0029] Table 1 Oligonucleotide sequence of sgRNA guide sequence

[0030]

Nucleotide sequence (5' to 3')

...

Embodiment 2

[0036] (1) Culture and cryopreservation of porcine fibroblasts

[0037] Put the pig fetus in a large domestic dish and add physiological saline to wash it 5-6 times. After washing, remove the fetal limbs, tail, head and internal organs. Put the processed fetus into an imported small dish, cut it into pieces with scissors, and terminate it until the largest tissue piece is 1mm3. Add 2ml of 0.25% EDTA-Trypsin to the small dish, place the small dish in a 37 degree incubator for 25 minutes to digest, then stop the digestion with 4ml of culture medium DMEM / F12 (10% FBS), blow it with a Pasteur pipette and suck out the liquid In a 15ml centrifuge tube, centrifuge at 1000rpm for 3min. After centrifugation, discard the supernatant, add 3ml of culture medium to resuspend, and divide evenly among three imported large dishes, and add 8ml of culture medium DMEM / F12 (10% FBS) to the imported large dishes. Observe the state of the cells the next day, and if the cell volume reaches 90%, pa...

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 provides an SgRNA (Small Guide Ribonucleic Acid) targeting sequence of specific target pig IRS1 (Insulin Receptor Substrate 1) gene and application thereof, and relates to the sgRNA targeting sequence and the application. The sgRNA targeting sequence is CGTAGTACTCGAGGCGCGCG. According to the sgRNA targeting sequence provided by the invention, the IRS1 gene can be knocked out or edited through a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-associated Protein) 9 system, so that expression of IRS1 can be removed, and the foundation is laid for preparing IRS1 transgenic pigs.

Description

technical field [0001] The invention relates to a sgRNA guide sequence and its application. Background technique [0002] IRS-1 is the main insulin receptor substrate, and through autophosphorylation, it transmits and radiates insulin signals to lower levels, thereby inducing the growth of individual systems and affecting glucose metabolism. IRS-1 is distributed in various tissue cells throughout the body, and is mainly expressed in skeletal muscle. Current studies suggest that the expression of IRS-1 may be closely related to patients with family history of type Ⅱ diabetes mellitus, and insufficient expression or abnormal phosphorylation of IRS-1 can lead to insulin resistance. The study by Katsutaro Morino et al. showed that in such diabetic patients, the phosphorylation level of Ser312 and Ser136 of IRS-1 increased by 50%, and the phosphorylation level of Akt decreased by 60%, which resulted in the glucose uptake of skeletal muscle cells in patients. 60% lower than the ...

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): C12N15/113C12N15/85C12N15/90
CPCC12N15/113C07K14/47C12N9/22C12N15/85C12N15/907C12N2810/10
Inventor 牟彦双翁晓刚刘忠华吕嘉伟郭佳
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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