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Establishment of CRISPR/Cas9-edited FGF5 Cell Strains in Cashmere Goat |
A Li ma, GAO Yuan, SU Xiao-hu, ZHOU Huan-min |
College of Life Science, Inner Mongolia Agricultural University, Key Laboratory of Bio-Manufacturing of Inner Mongolia Autonomous Region, Hohhot 010018, China |
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Abstract To establish CRISPR/Cas9-edited FGF5 cell strains, we designed gRNA targeted sites around the first extra of the FGF5 gene and constructed two vectors by Cas/gRNA plasmid construction kit weredesigned. vectors into cashmere goat fibroblasts by electroporation respectively was transfected. T7 endonuclease 1 (T7E1) was used for the detection of mutation efficiency. The best vector was transfected into cashmere goat fetal fibroblasts and all the monoclones was cultured. Then all the cell colonies by sequencing were idenfified. Sequencing results demonstrated that CRISPR/Cas9 was available for FGF5 gene edited and 20 FGF5+/- and FGF5-/- cell colonies were obtained, and the effiency was 14.81%. The double mutated cell colonies could be used as donor cells to construct reconstructed embryos which provide the possibility in production of FGF5 edited cashmere goat in the future.
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Received: 07 January 2016
Published: 02 March 2016
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[1] 王丙萍.靶除FGF5基因体细胞克隆绒山羊的研究.内蒙古:内蒙古农业大学,生命科学学院,2014.Wang B P.The Study on the Cloned Goat Knockout FGF5 Gene Transferred Somatic Cell.Inner Mongolia Agricultural University,College of Life Science,2014.
[2] Hebert J M,Rosenquist T,Gotz J,et al.FGF5 as a regulator of the hair growth cycle:evidence from targeted and spontaneous mutations.Cell,1994,78(6):1017-1025.
[3] Nguyen H Q,Danilenko D M,Bucay N,et al.Expression of keratinocyte growth factor in embryonic liver of transgenic mice causes changes in epithelial growth and differentiation resulting in polycystic kidneys and other organ malformations.Oncogene,1996,12(10):2109-2119.
[4] Rosenquist T A,Martin G R.Fibroblast growth factor signalling in the hair growth cycle:expression of the fibroblast growth factor receptor and ligand genes in the murine hair follicle.Dev Dyn,1996,205:379-386.
[5] Sundberg J P,Rourk M K,Boggess D,et al.Angora mouse mutation:altered hair cycle,follicular dystrophy,phenotypic maintenance of skin grafts,and changes in keratin expression.Vet Pathol,1997,34(5):171-179.
[6] 高爱琴,李宁,李金泉,赵兴波.山羊FGF5基因单核苷酸多态性群体遗传学分析.华北农学报,2006,21(3):71-76.Gao A Q,Li N,Li J Q,et al.Analysis on single nucleotide polymorphisms of FGF5 gene in different goat breeds.Acta Agriculturae Boreali-Sinica,2006,21(3):71-76.
[7] Lillestøl R,Redder P,Garrett R A,et al.A putative viral defence mechanism in archaeal cells.Archaea,2006,2(1):59-72.
[8] Bolotin A,Quinquis B,Sorokin A,et al.Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin.Microbiology,2005,151(8):2551-2561.
[9] Barrangou R,Fremaux C,Deveau H,et al.CRISPR provides acquired resistance against viruses in prokaryotes.Science,2007,315(5819):1709-1712.
[10] Jinek M,Chylinski K,Fonfara I,et al.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science,2012,337(6096):816-821.
[11] Sapranauskas R,Gasiunas G,Fremaux C,et al.The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli.Nucleic Acids Res,2011,39(21):9275-9272.
[12] Magadán A H,Dupuis M È,Villion M,et al.Cleavage of phage DNA by theStreptococcus thermophilus CRISPR3-Cas system.PLoS One,2012,7(7):e40913.
[13] Cong L,Ran F A,Cox D,et al.Multiplex genome engineering using CRISPR/Cas systems.Science,2013,339(6121):819-823.
[14] Mali P,Yang L,Esvelt KM,et al.RNA-guided human genome engineering via Cas9.Science,2013,339(6121):823-826.
[15] Ding Q,Regan Stephanie N,Xia Y,et al.Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs.Cell Stem Cell,2013,12(4):393-394.
[16] Ni W,Qiao J,Hu S,et al.Efficient gene knockout in goats using CRISPR/Cas9 system.PLoS One,2014,9(9):e106718.
[17] Feng Z Y,Zhang B T.Efficient genome editing in plants using a CRISPR/Cas system.Cell Res,2013,23(10):1229-1232.
[18] Hwang W Y,Fu Y,Reyon D,et a1.Efficient genome editing in zebrafish using a CRISPR-Cas system.Nat Biotechnol,2013,31(3):227-229.
[19] Jinek M,East A,Cheng A,et a1.RNA-programmed genome editing in human cells.Elife,2013,2:e00471.
[20] Wang H,Yang H,Shivalila C S,et a1.One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.Cell,2013,153(4):910-918.
[21] He X L,Yuan C,Chen Y L.Isolation,characterization,and expression analysis of FGF5 isoforms in Cashmere goat.Small Ruminant Research,2014,116(2~3):111-117.
[22] Ma K,Wang J,Shen B,et al.Efficient targeting of FATS at a common fragile site in mice through TALEN-mediated double-hit genome modification.Biotechnol Lett,2014,36(3):471-479.
[23] 李辉,施振旦.CRISPR/Cas9新型基因打靶系统的研究进展.江苏农业学报,2013,29(4):907-911.Li H,Shi Z D.Research progress of gene targeting technology of CRISPR/Cas9 system.Jiangsu J of Agr Sci,2013,29(4):907-911. |
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