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| A Double Fluorescence Screening Strategy to Enhance the Efficiency of Gene Targeting |
| CHEN Jian-wu1,2, REN Hong-yan2, HUA Wen-jun2, LIU Xi-mei2, QI Shi-jin2, ZHOU Li2, OU Yang-yan3, BI Yan-zhen2, YANG Ye1, ZHENG Xin-min2 |
1. College of Animal Sciences, Yangtze University, Jinzhou 434025, China;
2. Hubei Key Lab of Animal Embryo Engineering and Molecular Breeding, Institute of Animal Science and Veterinary, Hubei Academy of AgroSciences, Wuhan 430064, China;
3. College of Agriculture, Hubei Three Groges Vocational and Technical College, Yichang 443000, China |
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Abstract Gene targeting technology mediated by homologous recombination is a powerful tool for gene function analyze, because of its high accuracy and the mutation we introduced can be predicted.While in eukaryotes, the ratio of homologous recombination is too low to do further screening and identification. For this reason, establishing a high efficiency and widely used screening strategy becomes important. A new screening strategy which can dramatically improve the homologous recombination efficiency based on visual "double fluorescence" screening was established. The green fluorescent protein and neomycin gene were used as positive selection markers, and red fluorescent protein gene for negative selection marker. After screening by G418, the cell clones which just expressing the green fluorescent protein gene were selected and detected by transboundary PCR. The new strategy was used to detecting the gene targeting of pig MSTN gene in pig cells combined with CRISPR/Cas9 technology. In the two target sites T1 and T2, the targeting efficiency screening by "double establishing" reached up to 80.5% and 86.7%, which the was dramatically improved. This visual "double fluorescent" screening strategy established will become a useful tool for animal gene editing in the future.
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Received: 12 July 2016
Published: 25 January 2017
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Cite this article:
CHEN Jian-wu, REN Hong-yan, HUA Wen-jun, LIU Xi-mei, QI Shi-jin, ZHOU Li, OU Yang-yan, BI Yan-zhen, YANG Ye, ZHENG Xin-min. A Double Fluorescence Screening Strategy to Enhance the Efficiency of Gene Targeting. China Biotechnology, 2017, 37(1): 58-63.
URL:
https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20170109 OR https://manu60.magtech.com.cn/biotech/Y2017/V37/I1/58
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| [1] Thomas K R, Capecchi M R. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell, 1987, 51(3):503-512.
[2] Doetschman T, Gregg R G, Maeda N, et al. Targeted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature, 1987, 330(6148):576-578.
[3] Koller BH, Smithies O. Inactivating the beat 2-microglobin locus in mouse embryonic stem cells by homologous recombination. Proc Natl Acad Sci USA, 1989, 86:8932-8935.
[4] Mansour S L, Thomas K R, Deng C, et al. Introduction of a lacZ reporter gene into the mouse int-2 locus by homologous recombination. Proc Natl Acad Sci USA, 1990, 87(19):7688-7692.
[5] Kan Y, Ruis B, Lin S. The mechanism of gene targeting in human somatic cells. PLoS Genet, 2014, 10(4):e1004251.
[6] Mansour S L, Thomas K R, Capecchi M R. Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells:a general strategy for targeting mutations to non-selectable gene. Nature, 1988, 336(6197):348-352.
[7] Stanford W L, Cohn J B, Cordes S P. Gene-trap mutagenesis:past, present and beyond. Nat Rev Genet, 2001, 2(10):756-768.
[8] Shinta S, Kiyoe U, Miho K, et al. Construction and applications of exon-trapping gene-targeting vectors with a novel strategy for negative selection. BMC Res Notes, 2015, 8(1):278-287.
[9] Mussolino C, Cathomen T. RNA guides genome engineering. Nat Biotechnol, 2013, 31(3):208-209.
[10] Thomson A J, Mcwhir J. Biomedical and agricultural applications of animal transgenesis. Mol Biotechnol, 2004, 27(3):231-244.
[11] Friedel R H, Plump A, Lu X, et al. Gene targeting using a promoterless gene trap vector ("targeted trapping") is an efficient method to mutate a large fraction of genes. Proc Natl Acad Sci USA, 102(37):13188-13193.
[12] Kim Y G, Cha J, Chandrasegaran S. Hybrid restriction enzymes:zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci USA, 1996, 93(3):1156-1160.
[13] Li T, Huang S, Zhao X, et al. Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes. Nucleic acids Res, 2011, 39(14):6315-6325.
[14] Qi LS, Larson M H, Gilbert L A, et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 2013, 152(2):1173-1183.
[15] Gao F, Shen X Z, Jiang F, et al. DNA-guided genome editing using the natronobacterium gregoryi argonaute. Nature Biotechnology, 2016,34(7):768-713. |
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