sgRNA targeting sequence of specific target human ABCB1 gene and application

A kind of specific and genetic technology, applied in sgRNA guide sequences and application fields, to achieve the effect of solving the problem of multi-drug resistance

Active Publication Date: 2016-03-30
JINAN UNIVERSITY
View PDF3 Cites 49 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, CRISPR-Cas9 technology also has the disadvantage of context dependen...

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 targeting sequence of specific target human ABCB1 gene and application
  • sgRNA targeting sequence of specific target human ABCB1 gene and application
  • sgRNA targeting sequence of specific target human ABCB1 gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] (1) sgRNA design

[0038] According to the genome sequence of human ABCB1 gene (geneID:5343), two sgRNAs targeting human ABCB1 gene were designed. The 20nt oligonucleotide sgRNA guide sequence is: Sg1: 5'-TTGGACTGTCAGCTGCTGTC-3' (located in the eighth exon of gene ABCB1) and Sg2: 5'-GACTGTCAGCTGCTGTCGTC-3' (located in the fifth exon of gene ABCB1 exon)( figure 1 ); add CACCG to its 5' end to obtain a forward oligonucleotide (Forwardoligo); obtain its corresponding DNA complementary strand according to the guide sequence, and add AAAC to its 5' end to obtain a reverse oligonucleotide (Reverseoligo ). Synthesize the above-mentioned forward oligonucleotides and reverse oligonucleotides respectively, denature the Forwardoligo and Reverseoligo of the synthesized sgRNA oligonucleotides in pairs, and anneal; after annealing, a double strand connected to the expression vector lentiCRISPRv2 vector can be formed, At the same time, the designed gRNA target sequence was blasted ...

Embodiment 2

[0046] (1) Co-transfection of core plasmid and packaging plasmid pMD2.G and psPAX2 into 293T cells

[0047] Cultivate 293T cells until the confluence rate of 293T cells reaches 50% to 60%, 12 to 18 hours after seeding is the best time for transfection; replace fresh culture medium before transfection, add 3mL medium to a 60mm dish; The amount used is 4 μg of core plasmid (lentiCRISPRv2-hABCB1-Sg1 or lentiCRISPRv2-hABCB1-Sg2 prepared in Example 1, and lentiCRISPRv2 empty vector as a control), psPAX23 μg, pMD2.G1 μg, PEI 24 μL, supplemented with DMEM to a total volume of 200 μL , the order of addition is DMEM, PEI and plasmid DNA (core plasmid, pMD2.G and psPAX2); then stand at room temperature for 30 minutes to fully polymerize PEI and plasmid DNA, and add the transfection system dropwise to the above-mentioned cultured 293T cells after the polymerization In a small dish, shake gently and place in 37°C 5% CO 2 Continue culturing in the incubator;

[0048] (2) Virus harvest an...

Embodiment 3

[0061] Example 3 In vitro cell experiments to identify the knockout effect of the human gene ABCB1 on the cells after screening

[0062] (1) western blot

[0063] The p-gp protein expression levels of the cell lines HCT-8 / Vsg1, HCT-8 / Vsg2, KBV200sg1 and KBV200sg2 successfully knocked out of the ABCB1 gene in Example 2 were identified by westernblot experiments, and the cell line HCT- 8 / V and KBv200 were used as controls, in which the primary antibody was Anti-ABCB1 (MMSC-13131, purchased from santacruze), the secondary antibody was Anti-mouseIgG, HRP-linkedAntibody (Product No. 7076, cellsignaling), and the specific method was the conventional westernblot operation process .

[0064] The result is as Figure 4 As shown, in HCT-8 / V and KBv200 cells, there is a significant difference in the expression of p-gp between the cells transfected with sg1 and sg2 and the cells only transfected with the vector, and p-gp protein is almost not expressed in sg1 and sg2, which can be As 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 gene engineering applications and particularly relates to a sgRNA targeting sequence of a specific target human ABCB1 gene and an application. The sgRNA targeting sequence of the specific target human ABCB1 gene is Sg1 or Sg2; the nucleotide sequences of Sg1 and Sg2 are as follows: Sg1: 5'-TTGGACTGTCAGCTGCTGTC-3'; Sg2: 5'-GACTGTCAGCTGCTGTCGTC-3'. Two single-stranded oligo sequences are designed and synthesized according to the sgRNA targeting sequence, double strands are formed through annealing and then connected with a Cas9 carrier, sgRNA and a CRISPR system are introduced into a target cell through the Cas9 carrier, Cas9 protein can find a matched DNA sequence under the guidance of sgRNA, cutting is performed, and the ABCB1 gene is knocked out.

Description

technical field [0001] The invention belongs to the field of genetic engineering applications, in particular to a sgRNA guide sequence specifically targeting human ABCB1 gene and its application. Background technique [0002] CRISPR-Cas9 is an immune invasion system evolved during the evolution of bacteria and archaea to resist foreign invasion, including defense against invading viruses and foreign DNA. In the field of modern genetic engineering applications, it has become the three major genome editing tools alongside TALEN (transcription activator-like effector nuclease) and ZFN (zinc-finger nuclease) technologies. Compared with TALEN and ZFN technology, CRISPR-Cas9 technology has specific DNA recognition ability. In the second type of CRISPR system, Cas9 endonuclease cuts double-stranded DNA under the guidance of sgRNA, causing double-strand breaks in the genome. Repair instability produces non-specific recombination to generate repair errors (insertion or deletion), wh...

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): C12N15/113C12N15/867A61K31/7088A61P35/00
CPCC07K14/47C12N15/113C12N15/86C12N2740/15043C12N2800/107C12N2800/80
Inventor 石智杨阳邱建阁张文姬蒋起韦覃武明陈耀郑迪威魏梦宁
Owner JINAN UNIVERSITY
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