Method for knocking out pig GOT1 gene by CRISPR/Cas9 system

A gene and gene knockout technology, applied in the biological field, can solve the problem of less research, achieve high knockout efficiency, improve construction efficiency, and improve knockout efficiency.

Active Publication Date: 2019-11-19
SHANXI AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the research on GOT1 gene mainly focuses on mice, and there are relatively few studies on mammals such as pigs.

Method used

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  • Method for knocking out pig GOT1 gene by CRISPR/Cas9 system
  • Method for knocking out pig GOT1 gene by CRISPR/Cas9 system
  • Method for knocking out pig GOT1 gene by CRISPR/Cas9 system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Obtaining of sgRNA specifically targeting pig GOT1 gene

[0051] sgRNA-1 targets the sequence from position 518 to position 537 of the porcine GOT1 gene, sgRNA-2 targets the sequence from position 919 to position 938 of the porcine GOT1 gene, and sgRNA-3 targets the sequence from position 517 to position 536 of the porcine GOT1 gene sequence of bits. The sequences of sgRNA-1, sgRNA-2 and sgRNA-3 are as follows:

[0052] SEQ ID NO.1: sgRNA-1: 5'-CATTCGGTCCTATCGCTATT-3';

[0053] SEQ ID NO.2: sgRNA-2: 5'-AGAAGATCGTGCGAGTGACG-3';

[0054] SEQ ID NO.3: sgRNA-3: 5'-ACATTCGGTCCTATCGCTAT-3'.

[0055] In order to facilitate the subsequent connection with the carrier, the BsmBI restriction site sequence was added to both ends of the sgRNA, and the obtained double-stranded DNA sequence is shown in Table 1.

[0056] Table 1 The double-stranded DNA sequences corresponding to the three sgRNAs

[0057]

Embodiment 2

[0058] Example 2 Construction of a CRISPR / Cas9 gene knockout vector containing sgRNA specifically targeting pig GOT1 gene

[0059] This example provides a CRISPR / Cas9 gene knockout vector containing an sgRNA specifically targeting the porcine GOT1 gene and its construction method.

[0060] The construction method of the CRISPR / Cas9 gene knockout vector containing the sgRNA specifically targeting the pig GOT1 gene comprises the following steps:

[0061] 1. Construction of linearized vector

[0062] Recover and expand the strain carrying the pHS-CR054 carrier (the pHS-CR054 carrier map is shown in figure 1 As shown, purchased from Beijing Hopson Gene Technology Co., Ltd.), the plasmid was extracted according to the instructions of the OMEGA plasmid extraction kit.

[0063] The extracted product was digested with the restriction endonuclease BsmBI. The reaction system for the restriction endonuclease BsmBI was: 1 μL of restriction enzyme BsmBI, 500 ng of pHS-CR054 vector plasmi...

Embodiment 3

[0072] Example 3 Knockout and detection of pig GOT1 gene

[0073] This example provides a method for knocking out the pig GOT1 gene using the CRISPR / Cas9 gene knockout vectors GOT1_KO1, GOT1_KO2 and GOT1_KO3 constructed in Example 2 containing sgRNA specifically targeting the pig GOT1 gene and detection of the expression level of the pig GOT1 gene method.

[0074] 1. Knockout of porcine GOT1 gene

[0075] (1) Cell culture and collection

[0076] PK15 cells were revived with complete medium, and after three passages, transfection was performed after the cells entered the logarithmic growth phase. The day before transfection, 5×10 5 ~8×10 5 Cells were seeded into a 6-well plate, and 2 mL of antibiotic-free medium was added to each well, and transfection was performed when the cell density reached 60%-80%.

[0077] (2) Transfection

[0078] According to the requirements of Lipofectamine 2000, the CRISPR / Cas9 gene knockout vectors GOT1_KO1, GOT1_KO2 and GOT1_KO3 constructed ...

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Abstract

The invention relates to the field of biotechnology, and in particular, relates to a method for knocking out a pig GOT1 gene by a CRISPR/Cas9 system. The invention provides an sgRNA specifically targeting to the pig GOT1 gene, a CRISPR/Cas9 gene knock-out vector containing the sgRNA, a method for knocking out the pig GOT1 gene by the vector, and a detection method for the pig GOT1 gene. The CRISPR/Cas9 knockout vector provided by the invention can achieve high efficiency of the pig GOT1 gene knockout, significantly improve the construction efficiency of GOT1 gene knockout cells and GOT1 gene knockout pigs, and provide an effective method and basis for the function research and application of the pig GOT1 gene.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a sgRNA specifically targeting the pig GOT1 gene, a CRISPR / Cas9 gene knockout vector containing the sgRNA, a method for knocking out the pig GOT1 gene using the vector, and a method for knocking out the pig GOT1 gene Detection method. Background technique [0002] GOT1 is a pyridoxal phosphate (PLP)-dependent transaminase that participates in the production of aspartate (Asp) and α-ketoglutarate (α-KG) in the cytoplasmic matrix The reversible reaction between glutamate (Glu) and oxaloacetate (OAA) plays an important role in maintaining cell redox balance, promoting tumor cell proliferation, and regulating amino acid metabolism. [0003] At present, the research on GOT1 gene mainly focuses on mice, and the research on mammals such as pigs is relatively small. By using the CRISPR / Cas9 system to knock out the porcine GOT1 gene, studying the function of the porcine GOT1 gene in vitro a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/90C12N5/10C12Q1/6888A01K67/027
CPCA01K67/0276A01K2217/075A01K2227/108C07K14/47C12N15/113C12N15/85C12N15/907C12Q1/6888C12Q2600/158
Inventor 成志敏蔡春波李步高张宁芳张万锋郭晓红高鹏飞曹果清
Owner SHANXI AGRI UNIV
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