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A gene knockout and gene technology, applied in the field of genetic engineering and genetic modification, can solve the problems of gene function loss, gene frameshift mutation, etc.
Inactive Publication Date: 2016-12-07
CHINA AGRI UNIV
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Problems solved by technology
DNA double-strand breaks can be repaired in two ways: one is non-homologous end-joining repair (NHEJ), which produces a random type of insertion / deletion repair at the double-strand break, which may cause genetic damage. Frameshift mutations, resulting in loss of gene function
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Embodiment 1
[0038] Example 1, Construction of CRSIPR / Cas9 Targeting Vectors cas-1-3, cas-3-3
[0039] According to the action principle of CRISPR / Cas9, the sgRNA sequence is designed in the CDS region of the pig MC4R gene, such as figure 1 shown.
[0040] Select cas9 targets 1-3:
[0041] The sgRNA sequence is 5'-ttctggaaccgcagcaccta-3', and according to the principle of complementary base pairing, its reverse complementary sequence is 5'-taggtgctgcggttccagaa-3';
[0042] Select cas9 target 3-3:
[0043] The sgRNA sequence is 5'-gactttctccttacacagtc-3', and its reverse complementary sequence is 5'-gactgtgtaaggagaaagtc-3 according to the principle of complementary base pairing.
[0044]The backbone of the pX330 vector needs to be digested with BbsI, so the cohesive end of the BbsI restriction site needs to be added to the sgRNA sequence to facilitate its ligation into the backbone of the pX330 vector. Add the sgRNA sequence and its complement to the BbsI cohesive end.
[0045] a. Synt...
Embodiment 2
[0048] Embodiment 2, the massive culture of positive bacterial colony
[0049] a. Pick the positive monoclonal colony with correct sequencing, add it to 2-5ml ampicillin-resistant LB medium, and shake vigorously at 37°C and 300rpm for 8h in a shaker.
[0050] b. Add the initial cultured bacterial solution to 100ml ampicillin-resistant LB medium at a dilution ratio of 1 / 500-1 / 1000, shake vigorously at 37°C and 300rpm for 12h-16h in a shaker.
Embodiment 3
[0051] The endotoxin extraction of embodiment 3, cas-1-3, cas-3-3 plasmid
[0052] See the instructions for the EndoFree Plasmid Maxi Kit from QIAGEN. After measuring the concentration of the extracted plasmid, it was subpackaged and frozen for subsequent transfection of pig fetal fibroblasts.
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Abstract
The invention relates to the field of genetic engineering and genetic modification, in particular to a method for obtaining an MC4R gene knockout pig by using a CRISPR / Cas9 system for editing MC4R genes through a somatic cell nuclear transfer technology. sgRNA is designed by aiming at two sites (a 6133-6152bp sequence and a 7127-7146 bp sequence in a CDS region of the MC4R gene) in the CDS region of the pig MC4R gene for the first time; in addition, the CRISPR / Cas9 system is used for simultaneously cutting the two sites; the large segment deletion of the MC4R gene is realized; in addition, the gene knockout individual pig subjected to large segment deletion is obtained; a feasible method is provided for studying the pig MC4R gene.
Description
technical field [0001] The present invention relates to the field of genetic engineering and genetic modification, in particular to editing MC4R gene by using CRISPR / Cas9 system, and obtaining MC4R gene knockout pigs through somatic cell nuclear transfer technology. Background technique [0002] PVN is a key to appetite regulation and mainly expresses MC4R. In the PVN region, the activity of MC4R is regulated by hormones secreted by different neurons of the arcuate nucleus (such as the agonist α-MSH and the antagonist Agrp, etc.). MC4R is activated after binding with the ligand, and the extracellular signal is transmitted to the cell, activating adenylyl cyclase, and increasing the concentration of intracellular cAMP. Finally, it activates gene transcription regulated by cAMP response element (cAMP Response element, CRE), thereby regulating the substance metabolism and gene expression of cells. In addition to participating in the classic signal regulation pathway, MC4R als...
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