Please wait a minute...

中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2016, Vol. 36 Issue (7): 41-47    DOI: 10.13523/j.cb.20160707
技术与方法     
CRISPR/Cas9编辑绒山羊FGF5基因细胞株的建立
阿力玛, 高原, 苏小虎, 周欢敏
内蒙古农业大学生命科学学院 内蒙古自治区生物制造重点实验室 呼和浩特 010018
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
 全文: PDF(1079 KB)   HTML
摘要:

拟利用CRISPR/Cas9技术建立编辑FGF5基因的绒山羊细胞株。在FGF5基因的第一外显子设计靶点并合成gRNA靶点引物,构建2个编辑FGF5基因的Cas/gRNA真核表达质粒载体。电穿孔法转染绒山羊成纤维细胞后T7核酸内切酶(T7E1)检测载体活性,选择活性最高的载体转染细胞,单细胞接种并扩繁,提取基因组DNA,PCR及测序鉴定。经测序分析共获得20个FGF5基因敲除细胞株(包括FGF5+/-和FGF5-/-),总突变率为14.81%。双敲除突变细胞株可作为供体细胞进行重构胚构建,为创造高产绒性状的FGF5基因编辑绒山羊奠定基础。

关键词: 基因编辑FGF5CRISPR/Cas9绒山羊    
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.

Key words: CRISPR/Cas9    Cashmere goat    Gene editing    FGF5
收稿日期: 2016-01-07 出版日期: 2016-03-02
ZTFLH:  Q789  
基金资助:

“转基因生物新品种培育”国家科技重大专项资助项目(2014ZX08008-002)

通讯作者: 周欢敏     E-mail: huanminzhou@263.net
服务  
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章  
阿力玛
高原
苏小虎
周欢敏

引用本文:

阿力玛, 高原, 苏小虎, 周欢敏. CRISPR/Cas9编辑绒山羊FGF5基因细胞株的建立[J]. 中国生物工程杂志, 2016, 36(7): 41-47.

A Li ma, GAO Yuan, SU Xiao-hu, ZHOU Huan-min. Establishment of CRISPR/Cas9-edited FGF5 Cell Strains in Cashmere Goat. China Biotechnology, 2016, 36(7): 41-47.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20160707        https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I7/41

[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.

[1] 武秀知,王宏杰,祖尧. 斑马鱼hoxa1a基因调控颅面骨骼发育的功能研究*[J]. 中国生物工程杂志, 2021, 41(9): 20-26.
[2] 毕博,张宇,赵慧. 酵母杂交系统在CRISPR/Cas9基因编辑系统脱靶率研究中的应用*[J]. 中国生物工程杂志, 2021, 41(6): 27-37.
[3] 胡暄,王松,于学玲,张晓鹏. 不稳定EGFP细胞模型的构建及其在基因编辑体系评价中的应用*[J]. 中国生物工程杂志, 2021, 41(5): 17-26.
[4] 王艳梅,寇航,马梅,申玉玉,赵宝顶,路福平,黎明. 利用CRISPR-Cas9技术失活黑曲霉中果胶酶基因及突变株性能评价*[J]. 中国生物工程杂志, 2021, 41(5): 35-44.
[5] 冷燕,孙康泰,刘倩倩,蒲阿庆,李翔,万向元,魏珣. 全球基因编辑作物监管趋势研究[J]. 中国生物工程杂志, 2021, 41(12): 24-29.
[6] 郭洋,陈艳娟,刘怡辰,王海杰,王成稷,王珏,万颖寒,周宇,奚骏,沈如凌. Pd-1基因敲除小鼠构建及初步表型验证[J]. 中国生物工程杂志, 2021, 41(10): 1-11.
[7] 郭洋,万颖寒,王珏,龚慧,周宇,慈磊,万志鹏,孙瑞林,费俭,沈如凌. Toll样受体4(TLR4)基因剔除小鼠构建及初步表型分析[J]. 中国生物工程杂志, 2020, 40(6): 1-9.
[8] 樊斌,陈欢,宋婉莹,陈光,王刚. 乳酸菌基因改造技术研究进展 *[J]. 中国生物工程杂志, 2020, 40(6): 84-92.
[9] 黄胜, 严启滔, 熊仕琳, 彭弈骐, 赵蕊. 基于CRISPR/Cas9-SAM系统CHD5基因过表达慢病毒载体的构建及对膀胱癌T24细胞增殖,迁移和侵袭能力的影响 *[J]. 中国生物工程杂志, 2020, 40(3): 1-8.
[10] 雷海英,赵青松,白凤麟,宋慧芳,王志军. 利用CRISPR/Cas9鉴定玉米发育相关基因ZmCen*[J]. 中国生物工程杂志, 2020, 40(12): 49-57.
[11] 王玥,牟彦双,刘忠华. 基于CRISPR/Cas系统的单碱基编辑技术研究进展*[J]. 中国生物工程杂志, 2020, 40(12): 58-66.
[12] 王伟东,杜加茹,张运尚,樊剑鸣. CRISPR/Cas9在人病毒感染相关疾病治疗研究中的应用*[J]. 中国生物工程杂志, 2020, 40(12): 18-24.
[13] 何秀娟,胡凤枝,刘秋丽,刘玉萍,祝玲,郑文云. 乳腺癌细胞QSOX1的CRISPR/Cas9基因编辑及其对增殖侵袭的影响研究*[J]. 中国生物工程杂志, 2020, 40(11): 1-9.
[14] 王志敏,毕美玉,贺佳福,任炳旭,刘东军. CRISPR/Cas9系统的发展及其在动物基因编辑中的应用 *[J]. 中国生物工程杂志, 2020, 40(10): 43-50.
[15] 菅璐,黄映辉,梁天亚,王利敏,马洪涛,张婷,李丹阳,王明连. 利用CRISPR/Cas9技术建立敲除JAK2基因K562细胞系 *[J]. 中国生物工程杂志, 2019, 39(7): 39-47.