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A genetic modification method suitable for pigs

A genetic modification and gene technology, applied in the field of rapid and efficient genetic modification, can solve the problems of heavy workload, high operation requirements for technicians, and low efficiency, and achieve high transgenic efficiency, high editing success rate, simple and efficient operation Effect

Active Publication Date: 2022-05-13
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These two methods have the following disadvantages: 1) Microinjection is required, which requires high operating requirements for technicians, and at the same time, expensive microinjection equipment needs to be purchased, which makes it impossible for many laboratories to carry out experiments; 2) Only one can be edited at a time Embryos, low efficiency, heavy workload, usually somatic cell nuclear transfer needs to transfer thousands of recombinant embryos, microinjection needs to use hundreds of embryos, and the cost is high; 3) The success rate of editing is low

Method used

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  • A genetic modification method suitable for pigs
  • A genetic modification method suitable for pigs
  • A genetic modification method suitable for pigs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Using type 6 rAAV (rAAV, recombinant adeno-associated virus) to carry CRISPR / Cas9 gene editing tools to edit Bama miniature pigs

[0032] 1. Method

[0033] 1.1 Packaging of gene editing tools

[0034] Synthesize the forward and reverse strands of the target gene gRNA and anneal to form double-stranded DNA with BbsI-digested cohesive ends. The sequences of forward strand (SEQ ID NO.1) and reverse strand (SEQ ID NO.2) are as follows:

[0035] 5'-CACCGGCGATTGGTGACCAGATCC-3' (SEQ ID NO. 1);

[0036] 5'-AAACGGATCTGGTCACCAATCGCC-3' (SEQ ID NO. 2).

[0037] Then use T4 ligase to clone the aforementioned double-stranded DNA into the pX601 (Addgene 107055, which contains the Cas9 gene) plasmid vector after digestion with BbsI to obtain the pX601-gRNA plasmid vector; amplify the pX330 (Addgene42230) plasmid vector by PCR The Cbh promoter in the pX601-gRNA plasmid vector was digested with XbaI and AgeI, the 7630 fragment was recovered by cutting the gel, and the CMV...

Embodiment 2

[0052] Example 2 Using type 3 rAAV to carry the EGFP gene to construct EGFP transgenic pigs

[0053] 1. Method

[0054] 1.1 Construction of recombinant virus

[0055] The EGFP gene was codon optimized, the whole gene was synthesized, digested and cloned into the pAAV.CMV.MCS.SV40 adeno-associated virus vector with T4 ligase to obtain the pAAV.CMV.EGFP.SV40 vector, and the recombinant AAV was prepared after the vector was verified by sequencing Virus. 293 cells were co-transfected with the three plasmids pAAV.CMV.EGFP.SV40, pAAV2 / 6 and pAdΔF6. After 72 hours of transfection, the cells were lysed to collect the virus, the virus was purified by ultracentrifugation with iodixanol, and the virus titer was determined by quantitative qPCR.

[0056] Introduction of rAAV into fallopian tubes

[0057] With the 1.2 section of embodiment 1.

[0058] detection

[0059] (1) Fluorescence detection

[0060] Put the born piglets under ultraviolet light, observe whether there is green flu...

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Abstract

The present invention provides a fast and efficient gene modification method suitable for pigs, which is to package gene editing tools in type 3 or type 6 adeno-associated virus, obtain recombinant adeno-associated virus, and then inject the recombinant adeno-associated virus into the mother Pig fallopian tubes. Compared with the traditional somatic cell nuclear transfer method and embryo microinjection method, the method of the present invention is simple and efficient in operation, high in gene modification efficiency, and has great application prospects.

Description

technical field [0001] The invention belongs to the field of genetic modification of animals, and in particular relates to a fast and efficient genetic modification method suitable for pigs. Background technique [0002] For the purpose of improving the production performance of pigs, increasing the disease resistance of pigs, or using it as a culture carrier for medical organ transplantation, etc., the genetic modification technology suitable for pigs has gradually highlighted its application value. [0003] The genetic modification technology of pigs mainly includes two parts. The first part is the gene editing tool, specifically, a plasmid with gene editing function; the second part is the method of introducing the gene editing tool into the zygote (fertilized egg or embryo). [0004] Early gene editing tools, such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) technology tools, all had cumbersome operation steps and vector co...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/864C12N15/90C12N9/22A01K67/027
CPCC12N15/86C12N15/907C12N9/22A01K67/0276A01K67/0278C07K14/4705C12N2750/14143A01K2217/075A01K2227/108
Inventor 包骥杨光杨阳步宏高孟雨廖光能何宇婷张炳琪
Owner WEST CHINA HOSPITAL SICHUAN UNIV