Efficient and stable fixed-point integrated gene knock-in method based on pCAG-flox-neo vector

Abstract

The invention provides an efficient and stable fixed-point integrated gene knock-in method based on a pCAG-flox-neo vector. The method comprises the following specific steps: (1) carrying out double enzyme digestion on a pCAG-STOP2 vector; the synthesized fragment is connected to a pCAG-STOP2 carrier; (2) carrying out enzyme digestion on the synthesized carrier again, and then connecting the carrier into polyA-loxP to obtain a pCAA-flox-neo carrier; (3) inserting the sgRNA sequence of the pig pH 11 site capable of being identified by the sgRNA and the PAM sequence into the T3 promoter, so as to obtain a final gene knock-in vector skeleton pCAG-floxP-neo-pH 11, wherein the gene knock-in vector skeleton pCAG-floxP-neo-pH 11 is obtained; and (4) carrying out PCR (Polymerase Chain Reaction) amplification on the required hKCNH2-T618I and hmuPKD2 fragments, and inserting the fragments into a pCAG-floxP-neo-pH11 vector skeleton in a one-step cloning manner. The invention establishes a method which is efficient, accurate, simple to operate and capable of knocking out the target gene at the same time, and has important scientific significance for researching the effect of the target gene in mammals or disease models.

Description

technical field [0001] The invention relates to the technical field of gene editing, in particular to an efficient and stable site-directed integration gene knock-in method based on pCAG-flox-neo vector. Background technique [0002] As an experimental animal model, pigs are not only superior to rodents, but also have extremely high similarities with humans in terms of physiology and anatomy. As an animal model for researchers to study human diseases, genetically modified pigs have important clinical significance for the development of biomedicine and the improvement of human health. However, the production of genetically modified pigs is a lengthy and inefficient process that severely hampers the prospects for the development of genetically modified pig models. [0003] For example, in the site-directed integration experiment using IRES-IGF1-2A-Fat1, the integration efficiency of positive cell clones with dual-trait genes was 2.3% (Youwen Ni. Preparation of gene-edited pig...

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Problems solved by technology

[0005] In the study of using CRISPR / Cas9 technology to knock in the pig pRSAD2 gene, the efficiency of Rosa26-pRSAD2 site-specific knock-in was lower than 25% (Xie Z et al., Generation of pRSAD2 gene knock-in pig via CRISPR / Cas9technology.Antiviral Res.2020Feb; 174:104696);

[0006] In the studies of Li et al. using site-directed editing of pig genome and abnormal methylation of cloned pig DNA, the working efficiency of the plasmid was only about 45% (Li G, Jia Q, Zhao J, Li X, Yu M, Samuel MS, Zhao S, Prather RS, LiC. Dysregulation of genome-wide gene expression and DNA methylation inabnormal cloned piglets. BMC Genomics. 2014 Sep 24; 15(1): 811)

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