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Marking-free porcine beta-alexin 2 gene fixedpoint knock-in plasmid vector and application thereof

A marker-free, gene-based technology, applied in the field of genetic engineering of transgenic animals, which can solve problems such as the uncertainty of the safety of GMOs

Inactive Publication Date: 2020-04-03
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, traditional transgenic technologies such as microinjection, viral vectors, and transposon insertions can achieve random integration of target genes in the genome, but this random integration also brings uncertainty to the safety of genetically modified organisms. The existing problems include whether the copy number and insertion site of the gene insertion in the transgenic operation are clear, whether the expression of the exogenous gene is stable, whether it contains marker genes, etc.

Method used

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  • Marking-free porcine beta-alexin 2 gene fixedpoint knock-in plasmid vector and application thereof
  • Marking-free porcine beta-alexin 2 gene fixedpoint knock-in plasmid vector and application thereof
  • Marking-free porcine beta-alexin 2 gene fixedpoint knock-in plasmid vector and application thereof

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Experimental program
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Effect test

Embodiment 1

[0029] Embodiment 1: Construction of site-directed knock-in plasmid vector of pbd-2 gene

[0030] 1. Construction of pcCAG-R26-PBD2-T2A-PBD2 donor plasmid

[0031] Using the pcCAG-PBD2 vector (Yang et al.2015) as a template, the 5' end was amplified by extension PCR with KOD-Plus-Neo high-fidelity enzyme (Toyobo, Shanghai) and the EcoRI restriction site and Kozak sequence were added to the 3' end The pbd-2 gene with the BamHI restriction site added, and the pbd-2 gene with the BamHI restriction site and T2A sequence added at the 5' end and the XhoI restriction site added at the 3' end were amplified at the same time, and then the two The two pbd-2 genes were digested and connected by BamHI, and the fragment was named PBD2-T2A-PBD2.

[0032] The pcCAG-PBD2 vector and PBD2-T2A-PBD2 were digested with EcoRI and XhoI and then connected, and the recombinant vector obtained with the correct sequence was named pcCAG-PBD2-T2A-PBD2.

[0033] Taking the reference sequence (Genbank: NC...

Embodiment 2

[0043] Example 2: Construction and Identification of Marker-Free Rosa26 Gene Locus Transfected Large White Pig with pbd-2 Gene

[0044] 1. Construction of marker-free Rosa26 gene locus transfected with pbd-2 gene Large White pig

[0045] Firstly, marker-free Rosa26 gene locus transfected embryonic fibroblasts of pbd-2 gene were constructed, the procedure is shown in image 3 . When the confluence of porcine embryonic fibroblasts reaches about 80% in a 100mm diameter cell culture dish, discard the old medium, rinse twice with Duchenne's phosphate buffered solution (DPBS), add 1 mL of trypsin to digest until the cells detach , add a little complete medium (Dulman's modified Eagle medium + 10% fetal bovine serum + 1% penicillin-streptomycin) to stop the digestion, discard the supernatant after centrifugation and add DPBS to resuspend, and centrifuge again to discard the supernatant.

[0046] Add 800 μL of Gene Pulser Electroporation Buffer (Bio-Rad Life Medical Products Co., Lt...

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Abstract

The invention belongs to the technical field of animal gene engineering, and particularly relates to a marking-free porcine beta-alexin 2 gene fixedpoint knock-in plasmid vector and an application thereof. The marking-free porcine beta-alexin 2 gene fixedpoint knock-in plasmid vector disclosed by the invention is relevant to the field of transgenic animals. A donor plasmid pcCAG-R26-PBD2-T2A-PBD2which can express dual-copy pbd-2 genes is constructed, and the donor plasmid further contains a neomycin resistant gene neoR expression box having loxP sequences on two sides. Experiment certificatesthat when the plasmid and a targeting plasmid pX330-pRosa26 which can express Cas9 protein and guide RNA targeting a porcine Rosa26 site at the same time are electrically shifted into recipient cellsat the same time, fixedpoint integration of a pbd-2 gene into the porcine Rosa26 site is realized; through Cre recombinase, resistant marking is deleted; and through somatic nucleus transplantation and embryo transplantation, pbd-2 gene fixedpoint knock-in pigs free from resistant marking can be obtained.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering of transgenic animals, and in particular relates to a site-specific knock-in donor plasmid pcCAG-R26-PBD2-T2A-PBD2 for overexpressing porcine β-defensin 2 (pbd-2) gene and a construction method thereof. The recombinant plasmid of the present invention can be used to prepare cell lines without marker pbd-2 gene site-specific knock-in and marker-free pbd-2 gene site-specific knock-in transgenic pigs. Background technique [0002] Defensins are a class of cationic antimicrobial peptides, generally composed of 18-45 amino acids, containing 6 cysteine ​​residues, forming 3 intramolecular disulfide bonds. In vertebrates, defensins are divided into three categories: α, β and θ, according to their spatial structure and disulfide bond binding ability. So far, studies have shown that only beta defensins are present in pigs. Through sequence comparison with the known porcine β-defensin-1, porci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/66C12N15/90C12N5/10A01K67/027
CPCA01K67/0276A01K67/0278A01K2207/15A01K2217/072A01K2217/075A01K2227/108A01K2267/02C07K14/4723C12N15/66C12N15/85C12N15/907C12N2800/30
Inventor 周锐黎璐黄靖王安田黄超
Owner HUAZHONG AGRI UNIV
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