Method for porcine H11 site-specific insertion by using site specific cleavage system

A fixed-point cutting and locus technology, applied in the field of genetic engineering, can solve the problems of cumbersome steps, random insertion, and high price, and achieve the effect of simple cell screening method, high efficiency, and reduced cost

Active Publication Date: 2015-04-22
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] An object of the present invention is to provide a method for fixed-point insertion of pig H11 site by means of a fixed-

Method used

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  • Method for porcine H11 site-specific insertion by using site specific cleavage system
  • Method for porcine H11 site-specific insertion by using site specific cleavage system
  • Method for porcine H11 site-specific insertion by using site specific cleavage system

Examples

Experimental program
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Effect test

Embodiment 1

[0028] The method for inserting green fluorescent protein at the porcine H11 site by means of a site-specific cleavage system comprises the following steps:

[0029] 1. Targeting carrier construction

[0030] (1) Synthetic fragments

[0031] According to the DNA sequence of the porcine H11 site, the 3'-end homology arm (shown in sequence 3), the corresponding universal primer (shown in sequence 4) and restriction enzyme cutting sites were added at both ends: MluI (ACGCGT) and FseI ( GGCCGGCC) added, the synthetic fragment is as follows:

[0032]ACGCGTttcccgaggctGagttagttgGtccagccagtgattgagttgcgtgcggagggcttcttatcttagTTTTATAGGCTACACTGTTAACACTCAGGCTGTTTTCTACCGTTTAGTCAAAATATAGTCACCTTGCCTGCTTCACCTGTCCATCAGAGAATGGCCTCATTAATTGACTCTCTAGTATGAAGTCAAAGTAGCTTTGGTGGCCCTAAATGGACAAGTATCAAGAGACTGGGTGAATTGAGGAGCTTGAGACTGTCACCTCAGATCGAAAAGACTGAAAAATCACCTCAGATCAAAAAGACTGAAAAATCTTCAGTCTGGAAAGGGGACTCAAAACCATAATTAGAGTATTCTGGTAGAATCCTTTTCTCCACTGTTATTCATACAGTTAAGGTGAATAACTAAAAGTAATTGTGAGCTGAGGAGTAAG...

Embodiment 2

[0055] Use the same method as in Example 1 to construct a targeting vector that does not contain the gene sequence of the screening element DTA, co-transfect the PEF cells with the vector and the CRISPR / Cas9 system (the transfection system and method are the same as before), and culture at 37°C for 3 days later, filter by:

[0056] The screening method is the same as that with DTA, see Example 1 for specific operations. This method can also screen out positive clones, and a total of 192 clones were picked out, 104 of which were positive clones, with an efficiency of 54%. This experiment proved that positive clones could also be obtained without DTA.

[0057] Use the same method as in Example 1 to construct a targeting vector that does not contain the gene sequences of the screening elements DTA and Neo, and co-transfect PEF cells with the vector and the CRISPR / Cas9 system. Refer to Example 1 for the transfection system and specific methods. The DTA-free and Neo gene screening...

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Abstract

The invention provides a method for porcine H11 site-specific insertion by using a site specific cleavage system. The method comprises the following steps: 1) designing5'-terminal homologous arm and 3'-terminal homologous arm of knocked-out gene and corresponding universal primers; 2) introducing the above homologous arms, the universal primers and gene to be inserted into a vector in order to a targeting vector; 3) transferring into cells; and 4) carrying out PCR amplification, and identifying the insertion result. The targeting vector is designed mainly relying on the porcine H11 site cleavage system, and the vector can accurately introduce exogenous gene into the porcine the H11 site in order to solve the problems of low efficiency of traditional targeting techniques, inconvenient designing of PCR detection primers and large detection difficulty. The site-specific insertion method provided by the invention allows the exogenous gene to be stably expressed in H11, and builds a stable platform for pig transgene.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a fixed-point insertion method for porcine H11 sites by using a fixed-point cutting system and a designed targeting carrier. Background technique [0002] It is known that in biotechnology research, the method of homologous recombination or transposon can be used to insert the target gene into the chromosomal genome, but practice shows that the efficiency of homologous recombination is low, the operation is troublesome, and due to the purpose The original gene is destroyed by the insertion of the gene; the method of using the transposon also has the fact that the position inserted into the chromosome is random, and the operation transposase used is expensive. [0003] Therefore, due to the limitations of the above-mentioned techniques, when cultivating good breeds of pigs, the method of random integration is mainly used to randomly insert foreign genes int...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/63C40B50/06
Inventor 李奎阮进学杨述林李和刚牟玉莲吴添文魏景亮徐奎黄雷周荣刘楠
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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