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.
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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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