Production method for Fbxo40 gene knockout pigs

A gene knockout and gene technology, applied in the field of animal genetic engineering and genetic modification, can solve the problems of gene function loss, gene frameshift mutation, etc.

Inactive Publication Date: 2016-08-03
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 i...

Method used

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  • Production method for Fbxo40 gene knockout pigs
  • Production method for Fbxo40 gene knockout pigs
  • Production method for Fbxo40 gene knockout pigs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Construction of CRSIPR / Cas9 Targeting Vector pX3301-8

[0031] 1. Design and screening of porcine Fbxo40 gene-specific sgRNA

[0032] The sequences of exon 3 and exon 4 of porcine Fbxo40 gene encode important domains of Fbxo40 protein—zinc-finger domain and F-box domain.

[0033]According to the working principle of CRISPR / Cas9, a total of 17 sgRNA sequences were designed at different positions of the third exon and the fourth exon. Its sequence and complementary sequence are shown in Table 1.

[0034] Table 1

[0035]

[0036]

[0037] The bold part in Table 1 is the PAM sequence recognized by CRISPR / Cas9.

[0038] 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.

[0039] The designed sgRNA and its complementary sequence added to the cohesive end of the BbsI restriction site were s...

Embodiment 2

[0052] Example 2 Establishment of pig fetal fibroblasts

[0053] Anesthetize the 30-day-pregnant piglet, aseptically remove the fetus from the uterus, wash the fetus with PBS containing double antibodies, place it in an ultra-clean workbench, and remove the head, limbs, internal organs and cartilage of the fetus with ophthalmic scissors Rinse the tissue with PBS; use ophthalmic scissors in the cell culture dish to cut the remaining tissue into about 1mm 3 Small pieces; add an appropriate amount of FBS to keep the tissue from drying out too much. Transfer the shredded tissue pieces to a T75 cell culture flask, and spread the tissue pieces evenly; add 5 mL of cell culture medium, with the side covered with the tissue pieces facing up, without being submerged by the culture medium, store at 37°C, 5% CO 2 After culturing in the incubator for 3 to 5 hours, turn the T75 over so that the tissue block is submerged in the culture medium; after culturing for about 3 days, it is observ...

Embodiment 3

[0054] Example 3 Transfection of pig fetal fibroblasts and screening of target cell monoclonal

[0055] 1. Digestion of PGK-Neo plasmid

[0056] The PGK-Neo plasmid was digested with restriction endonucleases NotI and NheI from NEB Company, and kept in a water bath at 37°C for 4h. The PGK-Neo fragment with a size of 1.8 kb was recovered using Genstar's Gel Extraction Kit. After the concentration was measured, it was subpackaged and frozen in a -20°C refrigerator for later use.

[0057] 2. Digest and centrifuge porcine fetal fibroblasts that have reached 80-90% confluence in a six-well plate, and obtain about 2×10 5 -2×10 6 porcine fetal fibroblasts.

[0058] 3. Add 2 μg of the CRISPR / Cas9 targeting vector pX3301-8, and add it to the Lonza transfection reagent at a molar ratio of 3:1 with the PGK-Neo gene linear vector, and mix well. The cells were resuspended using the transfection reagent added with the plasmid, and the cell suspension was added to the electric shock cup...

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Abstract

The invention provides a production method for Fbxo40 gene knockout pigs. The production method comprises the steps that a CRISPR-Cas9 targeting vector and a PGK-Neo resistant gene are jointly transfected into a porcine embryonic fibroblast to obtain a G418-resistant positive monoclonal cell, an Fbxo40 gene in the positive monoclonal cell generates insertion or deletion mutation, a reading frame generates frame shift and stops in advance, then the obtained cell is cloned to serve as a donor cell for nuclear transfer, and an oocyte serves as a receptor oocyte for nuclear transfer; cloned embryos are obtained through a somatic cell nuclear transfer technique, the high-quality cloned embryos are transferred into the fallopian tube of an oestrous sow, and the Fbxo40 gene knockout pigs are obtained through whole-period development. According to the production method, the Fbxo40 knockout pigs are efficiently obtained through a CRISPR-Cas9 gene editing technique at the low cost, and animal models are supplied to research of muscle development and diseases related to the muscles.

Description

technical field [0001] The invention belongs to the field of animal genetic engineering and genetic modification, and specifically relates to a method of editing pig Fbxo40 gene by using CRISPR / Cas9 system, and obtaining Fbxo40 gene knockout pig by somatic cell nuclear transfer technology. Background technique [0002] IGF1 can lead to muscle hypertrophy by activating the IGF1R / IRS1 / PI3K / AKT pathway, but in differentiated muscle cells, the substrate IRS1 of IGF1 will be ubiquitinated and degraded by the proteasome, resulting in the interruption of the IGF1 signaling pathway . The study found that the E3 ubiquitin ligase Fbxo40 mediates the rapid conversion of IRS1. The Fbxo40-SCF-E3 protein complex directly ubiquitinates IRS1, and this ubiquitination is enhanced when IRS1 is phosphorylated on tyrosine. After knocking out Fbxo40 in mice, muscle fibers were significantly thickened. During the rapid growth period of mice, IGF1 is highly expressed. After Fbxo40 knockout, the ...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/113C12N15/85C12N15/873A01K67/027
CPCA01K67/0276A01K2207/10A01K2217/075A01K2227/108A01K2267/03C07K14/47C12N15/113C12N15/8509C12N15/8778C12N2310/10C12N2800/107C12N2800/80C12N2810/10
Inventor 李秋艳邹云龙李宁赵要风李志远付怡静郝海阳
Owner CHINA AGRI UNIV
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