Knockout vector, targeting vector, in-vitro knockout method of PPARG gene in liver cell, and knockout liver cell

A technology for knocking out vectors and liver cells, which is applied in the field of molecular biology, can solve the problems of low knockout efficiency and cumbersome knockout process, and achieve the effects of high knockout efficiency, less off-target, and convenient construction and use

Inactive Publication Date: 2019-02-01
CHINA NAT CENT FOR FOOD SAFETY RISK ASSESSMENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current in vitro knockout methods for hepatocyte PPARG gene, such as RNA interference, zinc finger nuclease, transcription activator-like effector nuclease, etc., are cumbersome and have low knockout efficiency.

Method used

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  • Knockout vector, targeting vector, in-vitro knockout method of PPARG gene in liver cell, and knockout liver cell
  • Knockout vector, targeting vector, in-vitro knockout method of PPARG gene in liver cell, and knockout liver cell
  • Knockout vector, targeting vector, in-vitro knockout method of PPARG gene in liver cell, and knockout liver cell

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preparation example Construction

[0047] The embodiment of the present invention also provides a preparation method of the knockout vector, comprising the following steps:

[0048] S11, annealing the complementary DNA sequence to form a double-stranded oligonucleotide fragment;

[0049] S12, providing the CRISPR / Cas9 carrier backbone, the sequence of the cohesive end of the CRISPR / Cas9 carrier backbone can be complementary to the sequence of the cohesive end of the double-stranded oligonucleotide fragment; and

[0050] S13, connecting the CRISPR / Cas9 vector backbone and the double-stranded oligonucleotide fragment to form a closed circular plasmid.

[0051] In step S11, the double-stranded oligonucleotide fragment can be formed by annealing two single strands of the complementary DNA sequence. The annealing condition may be that the mixed aqueous solution of two single chains is placed in a water bath at a temperature of 95° C. to 100° C. for 3 minutes to 6 minutes, and then naturally cooled to room temperatu...

Embodiment

[0096] Preparation of knockout vectors:

[0097] Select SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 of the PPARG gene in HL-7702 cells (human liver normal cells) These 6 specific sequences were used as the target sequences for knockout.

[0098] Provide such as SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, Single-stranded DNA sequences of SEQ ID NO.15 and SEQ ID NO.16 and SEQ ID NO.17 and SEQ ID NO.18.

[0099] The 12 single-stranded DNA sequences were respectively dissolved in pure water to adjust the final concentration to 100 μM.

[0100] Annealing: SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, 15 μL of each of SEQ ID NO.15 and SEQ ID NO.16, and SEQ ID NO.17 and SEQ ID NO.18 were mixed, put into a boiling water bath for 5 minutes, and then cooled to room temperature naturally to form 6 co...

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Abstract

The present invention discloses a knockout vector. The knockout vector comprises a CRISPR / Cas9 vector skeleton and a segment of a complementary DNA sequence linked to the CRISPR / Cas9 vector skeleton;the CRISPR / Cas9 vector skeleton can express Cas9 nuclease; the complementary DNA sequence can be correspondingly transcribed into sgRNA capable of specifically recognizing a specific sequence of in aPPARG gene, the sgRNA can be complementarily paired with a complementary strand of a corresponding specific sequence in the PPARG gene to make the Cas9 enzyme specifically cleave the specific sequence, and the specific sequence includes one or more of sequences represented by SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6. The invention also discloses a targeting vector. The targeting vector comprises a 5' homologous gene, a marker gene and a 3' homologous gene which are sequentially linked, and the 5' homologous gene and the 3' homologous gene are respectivelyhomologous with an upstream end contiguous gene and a downstream end contiguous gene of a cleavage site of the PPARG gene of a chromosome containing the PPARG gene in the liver cell. The invention further discloses an in-vitro knockout method of the PPARG gene in the liver cell, and a PPARG gene knockout liver cell.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a knockout vector, a targeting vector, an in vitro knockout method of the PPARG gene in liver cells and a PPARG gene knockout liver cell. Background technique [0002] PPARG gene is the coding gene of peroxisome proliferative activated receptor gamma (peroxisome proliferative activated receptor, gamma), which belongs to the nuclear receptor peroxisome proliferative activated receptor subfamily, and can bind to retinoid receptor (RXR) Combine to form heterodimers, and then regulate the transcription of various genes by binding to specific DNA sequences. PPARG is mainly responsible for the catabolism of lipids, and is also associated with a variety of diseases, including: obesity, diabetes, arteriosclerosis, and cancer. Based on the current research and understanding of the regulatory mechanism of energy metabolism in the body, the academic community usually uses glucose and lipid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N9/22C12N5/10
CPCC12N9/22C12N15/907
Inventor 贾旭东杨辉张倩男
Owner CHINA NAT CENT FOR FOOD SAFETY RISK ASSESSMENT
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