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A method to increase the repair frequency of homologous recombination in sheep embryonic fibroblasts after gene editing
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A fibroblast and gene editing technology, applied in the biological field, can solve the problems of low gene targeting efficiency and low homologous recombination probability.
Inactive Publication Date: 2018-11-13
XINJIANG ACADEMY OF AGRI & RECLAMATION SCI
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Problems solved by technology
The probability of homologous recombination in normal mammalian cells is extremely low, resulting in extremely low gene targeting efficiency (10 -6 )
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Embodiment 1
[0041] Example 1. Screening of siRNAs Interfering with Lig4 Gene Expression in Sheep Embryo Fibroblasts
[0042] 1. Construction of eukaryotic expression plasmid pcDNA3.1-Shlig4
[0043] 1. Primer design
[0044] Primers were designed according to the predicted sheep Lig4 gene sequence (XM_004012236) and GAPDH gene sequence (NM_001289746.1) in Genbank, and were synthesized by Shanghai Sangon Biotechnology Co., Ltd. The primer sequences are listed in Table 1.
[0045] Table 1 is the primer name and sequence
[0046]
[0047] The underlines are the restriction sites BamHI and XbaI, and the boldfaces are the protective bases.
[0048] 2. Construction of pcDNA3.1-Shlig4 recombinant plasmid
[0049] According to the cDNA sequence of the mammalian Lig4 gene without introns, use the primers Shlig4F / Shlig4R to directly use the sheep genomic DNA as a template for PCR amplification, and obtain a 2736bp PCR product that matches the expected size, which is the cDNA of the sheep Lig...
Embodiment 2
[0074] Example 2, Improving the frequency of homologous recombination repair of sheep embryonic fibroblast genomic DNA
[0075] Lig4 gene siRNA improves HR repair efficiency in isolated sheep embryonic fibroblasts, as follows:
[0076] Due to the limited passage capacity of sheep primary fibroblasts, it is basically not feasible to establish stable clones. To quantify DNA end joining efficiency and fidelity, a plasmid rejoining assay was used.
[0077] The HR plasmid donated by Gorbunova's laboratory uses GFP (Green Fluorescent Protein) as a recombinant reporter gene. The reporter system contains 2 copies of mutated GFP-Pem1. In the first GFP-Pem 1 gene fragment, the first exon of GFP contains a deletion of 22 bp base length, and two reverse symmetric I-SceI enzyme recognition sites are inserted. The second GFP-Pem1 gene fragment lacks the promoter, ATG start codon and exons of the second part of GFP. When the I-SceI site is broken, non-complementary cohesive-terminal DSBs...
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Abstract
The invention discloses a method for increasing HR (Homologous Recombination) repairing frequency of ovine embryo fibroblasts after gene editing. The method provides application of a substance for restraining the expression of an Lig4 gene in in-vitro ovine embryo fibroblasts containing gene editing DNA (Deoxyribose Nucleic Acid) fragments in preparation of products for promoting HR repairing of the in-vitro ovine embryo fibroblasts containing the gene editing DNA fragments. An experiment of the invention shows that siRNA (Small Interfering Ribose nucleic Acid) which restrains the expression of the ovine Lig4 gene is screened, the expression of the ovine Lig4 gene is restrained through the siRNA, an intracellular HR repairing path is stimulated by restraining an NHEJ (Non-homologous End Joining) repairing path of the ovine embryo fibroblasts, the frequency of adopting HR repairing by cells is increased, and a basis is provided for increasing gene targeting efficiency of the ovine embryo fibroblasts and researching accurate editing of an ovine genome.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to a method for increasing the frequency of homologous recombination repair in sheep embryonic fibroblasts after gene editing. Background technique [0002] Using artificial endonucleases (Engineered endonuclease, EEN), such as zinc finger enzymes (Zinc fingernucleases, ZFNs), transcription activator-like effector nucleases (Transcription activator-like effector nucleases, TALENs) and CRISPR / Cas9 (Clustered regularly interspaced shortpalindromic Repeats (CRISPR) / CRISPR-associated 9 (Cas9)), site-directed cutting of the genome, generating double-strand breaks (Double-strand brakes, DSBs) or Nicks (Nicks) at specific sites, and promoting research on precise editing of mammalian genomes. [0003] Cells mainly repair DSBs through non-homologous end joining (NHEJ) pathway, resulting in gene knockout phenotype. Co-transfect EEN and single-stranded oligonucleotides, or donor templates of homo...
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