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CNE10 Gene Knockout in Epidermal Stem Cells Using CRISPR-Cas System

A technology for epidermal stem cells and cells, which is applied in the field of establishing epidermal stem cell lines, can solve the problem that siRNA cannot stabilize heredity, etc., and achieve the effects of high knockout efficiency, good knockout effect, and stable passage.

Active Publication Date: 2019-07-23
江西汉氏联合干细胞科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a kind of epidermal stem cell with CNE10 gene knockout, which effectively overcomes the technical defect that the prior art uses siRNA for interference and cannot stably inherit

Method used

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  • CNE10 Gene Knockout in Epidermal Stem Cells Using CRISPR-Cas System

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1. Construction of CRISPR expression vector

[0023] gRNA design

[0024] According to the gene sequence of the target gene, through the applicant's unique optimization design method, the specific form of sgRNA obtained by specific screening is as follows:

[0025] CNE10-sgRNA1: 5’to 3’gacgtcggattccagcctcc

[0026] CNE10-sgRNA2: 5’to 3’ccagcgcctggggctctccg

[0027] According to the above gRNA, CACC is added to the 5'end to obtain the forward oligonucleotide sequence, and AAAC is added to the 5'end of the complementary strand to obtain the reverse oligonucleotide sequence. The forward and reverse oligos are synthesized respectively. Nucleotide sequence, and then the synthesized sequence is denatured and annealed to obtain a double-stranded DNA fragment with BbsI sticky ends, as follows: Forward: 5'-CACCNNNNNNNNNNNNNNNNNNNNNNNN Reverse: NNNNNNNNNNNNNNNNNNNNCAAA-5', denaturation and annealing system: 2μl positive To the oligonucleotide chain (50μM) 2μl reverse oligonucleot...

Embodiment 2

[0031] Example 2 Cloning of the enhanced protein ESCS-higher and construction of the vector

[0032] The ESCS-higher gene was cloned, and the gene sequence described in SEQ ID NO:1 was obtained through the method of full gene synthesis. Using this sequence as a template, the sequence of the upstream and downstream primers was 5'-atgatatactttattagaat-3', 5 '-tcaagggatttccatttctc-3', primers and whole genome were synthesized by Shanghai Shenggong Co., Ltd. The PCR reaction amplifies the target gene fragment of the ESCS-higher gene. The amplification reaction system is as follows: 95°C, 40s, 57°C, 1min, 72°C, 1min, 72°C, 10min, cycle 35 times, PCR products are produced by Shanghai Shenggong Co., Ltd. Sequencing was performed, and the binding completely matched SEQ ID NO:1 through sequencing. Subsequently, the target gene amplified by PCR was connected to the empty vector lentiviral vector pHIV-CS-CDF-CG-PRE, and the recombinant lentiviral vector was identified by methods such as PC...

Embodiment 3

[0033] Example 3 Application analysis of CRISPR / Cas9 in epidermal stem cells

[0034] The sgRNA expression plasmid prepared in Example 1 and the well-known Cas9 expression plasmid were co-transfected into epidermal stem cells. Using liposome transfection method, the constructed transfection system of transfected epidermal stem cells and reagents make Lipofectamine TM 2000 (invitrogen company), refer to the transfection instructions for detailed transfection steps. Stem cells not transfected with the synergistic gene of Example 2 were used as a positive control.

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Abstract

The invention provides CNE10 gene editing for epidermal stem cells by using a CRISPR-Cas system, and particularly relates to establishment of an epidermal stem cell system with GNE10 gene knocked out.Through the establishment, two specific gRNA (guide ribose nucleic acids) are obtained, editing efficiency of the CRISPR-Cas system aiming at the GNE10 gene in the epidermal stem cells can be increased remarkably. An obtained epidermal stem cell plasmid with CNE10 gene knockout is good in hereditary stability and high in targeting efficiency.

Description

Technical field [0001] The present invention provides a CNE10 gene editing for epidermal stem cells by using a CRISPR-cas system, and particularly relates to the establishment of an epidermal stem cell line with CNE10 gene knockout. Background technique [0002] Epidermal stem cells (EpiSCS) are stem cells with self-proliferation and multidirectional differentiation potential. Their normal proliferation and differentiation are the basic requirements for maintaining the structural and functional integrity of the skin and its appendages (sweat glands, hairs, sebaceous glands). Under physiological conditions, epidermal stem cells differentiate into a stem cell and a transient expansion cell (transit amplifying cells) through asymmetric division. TA cells then differentiate into post-mitotic cells (Post-mitotic cells) and terminal after multiple divisions. Differentiated cells (terminally-differentiated cells) to supplement the need for continuous renewal of epidermal cells. Studies...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/90C12N9/22
CPCC12N9/22C12N15/113C12N15/907C12N2310/10
Inventor 韩之波杨骏朱成光
Owner 江西汉氏联合干细胞科技有限公司
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