CRISPR/Cas9介导获得用于分离双峰驼iPSCs的转染细胞系
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摘要
双峰驼(Camelus bactrianus)作为西北地区的濒危物种,其生理特性特殊,用途广泛。利用诱导性多功能干细胞(induced pluripotent stem cells, iPSCs)对该物种的种质资源进行保护具有重要的应用价值。但传统分离鉴定iPSCs的方法较为复杂繁琐。为了对iPSCs进行快速高效的分离鉴定,本实验利用CRISPR/Cas9系统,参照双峰驼Nanog基因,在其终止密码子附近设计单链导向RNA (single guide RNA,sgRNA),成功构建了切割载体;以双峰驼全基因组为模板获得上、下游同源臂,并在两个同源臂之间连入两个报告基因绿色荧光蛋白基因(green fluorescent protein, GFP)和新霉素抗性基因(neomycin resistance,Neo),成功构建了表达载体。同时测得双峰驼胎儿成纤维细胞对于遗传霉素(geneticin, G418)的最小致死量。用表达载体与切割载体共转染双峰驼胎儿成纤维细胞,继续培养48 h后,用G418筛选两周;之后用不含G418的DMEM/F12完全培养基对药筛后的细胞进行扩大培养,成功获得了活力较高的稳定转染细胞系。本实验为验证在Nanog基因后定点敲入报告基因来正确筛选iPSCs提供了生物材料。
Bactrian camels(Camelus bactrianus) is a endangered species in China's northwest region. It has special physiological characteristics and has widely uses. Induced pluripotent stem cells(iPSCs) has important application value in protecting the germplasm resources of this species. However, the traditional methods for the isolation and identification of iPSCs are complicated and cumbersome. For the rapid and efficient isolation and identification of iPSCs, the CRISPR/Cas9 system was used in this experiment. The single guide RNA(sgRNA) was designed near the stop codon in reference to the Nanog gene expressed only in the embryonic stem cells(ESCs) stage of Bactrian camel, and the cleavage vector was successfully constructed and screened;the whole camel genome was used as a template to obtain upstream and downstream homology arms, and two reporter genes of green fluorescent protein gene(GFP) and neomycin resistance gene(Neo) were insertedbetween the two homologous arms, the expression vector was successfully constructed. At the same time, the minimum lethal dose of geneticin(G418) of Bactrian camel fibroblasts was obtained by the minimum lethal dose experiment. After sequencing and verification of the above two vectors, the Bactrian camel fibroblasts were co-transfected with the expression vector and the cleavage vector and cultured for 48 h, and then cultured for two weeks in a selection medium containing G418; then, the normal culture medium was used to expand the cells after the drug screening, the highly viable stable transfected cell line was successfully obtained. This experiment will provide biomaterials for the correct screening of iPSCs by sequencing the reporter gene after the Nanog gene.
Bactrian camels(Camelus bactrianus) is a endangered species in China's northwest region. It has special physiological characteristics and has widely uses. Induced pluripotent stem cells(iPSCs) has important application value in protecting the germplasm resources of this species. However, the traditional methods for the isolation and identification of iPSCs are complicated and cumbersome. For the rapid and efficient isolation and identification of iPSCs, the CRISPR/Cas9 system was used in this experiment. The single guide RNA(sgRNA) was designed near the stop codon in reference to the Nanog gene expressed only in the embryonic stem cells(ESCs) stage of Bactrian camel, and the cleavage vector was successfully constructed and screened;the whole camel genome was used as a template to obtain upstream and downstream homology arms, and two reporter genes of green fluorescent protein gene(GFP) and neomycin resistance gene(Neo) were insertedbetween the two homologous arms, the expression vector was successfully constructed. At the same time, the minimum lethal dose of geneticin(G418) of Bactrian camel fibroblasts was obtained by the minimum lethal dose experiment. After sequencing and verification of the above two vectors, the Bactrian camel fibroblasts were co-transfected with the expression vector and the cleavage vector and cultured for 48 h, and then cultured for two weeks in a selection medium containing G418; then, the normal culture medium was used to expand the cells after the drug screening, the highly viable stable transfected cell line was successfully obtained. This experiment will provide biomaterials for the correct screening of iPSCs by sequencing the reporter gene after the Nanog gene.
引文
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Peters J,Driesch A.1997.The two humped camel(Camelusbactrianus):New light on its distribution management and medical treatment in the past[J].Journal of Zoology,242:651-679.
Plessis A,Perrin A,HaberJ,et al.1992.Site-specific recombination determined by I-Scel,a mitochondrial group I intron-encoded endonuclease expressed in the yeast nucleus[J].Genetics,130(3):451-460.
Rouet P,Smih F,Jasin M.1994.Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease[J].Molecular and Cellular Biology,14(12):8096-8106.
Tada M,Takahama Y,Abe K,et al.2001.Nuclear reprogramming of somatic cells by in vitro hybridization with EScells[J].Current Biology,11(19):1553-1558.
Takahashi K,Yamanaka S.2006.Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[J].Cell,126(4):663-676.
Takahashi K,Tanabe K,Ohnuki M,et al.2007.Induction of pluripotent stem cells from adult human fibroblasts by defined factors[J].Cell,131(5):861-872.
Wilmut I,Schnieke A E,McWhir J,et al.1997.Viable offspring derived from fetal and adult mammalian cells[J].Nature,385(6619):810-813.
Yamagchi S,Kimura H,Tada M,et al.2005.Nanog expression in mouse germ cell development[J].Gene Expression Patterns,5(5):639-646.
田净净,刘洋洋,杨哲,等.2016.利用CRISPR/Cas9系统高效敲除斑马鱼lncRNA基因启动子区[J].农业生物技术学报,24(5):649-656.(Tian J J,Liu Y Y,Yang Z,et al.2016.Efficient knockout of lncRNA promoter region by CRISPR/Cas9 system in zebrafish(Danio rerio)[J].Journal of Agricultural Biotechnology,24(5):649-656.)
王强,陈美珏,任兆瑞,等.2003.山羊β酪蛋白基因克隆及序列分析[J].中国生物化学与分子生物学报,19(1):17-23.(Wang Q,Chen M J,Ren Z R,et al.2003.Molecular cloning and sequence analysis of goatβ-Casein gene[J].Chinese Journal of Biochemistry and Molecular Biology,19(1):17-23.)
章静波.2002.组织和细胞培养技术[M].北京:人民卫生出版社,pp.119-124.(Zhang J B.2002.Tissue and Cel Culture Techniques[M].People's Medical Publishing House,Beijing,China,pp.119-124.)
Byrne J A,Pedersen D A,Clepper L L,et al.2007.Producing primate embryonic stem cells by somatic cell nuclear transfer[J].Nature,450(7169):497-502.
Chalfie M,Tu Y,Euskirchen G,et al.1994.Green fluorescen protein as a marker for gene expression[J].Science,263(5148):802-805.
Chambers I,Colby D,Robertson M,et al.2003.Functiona expression cloning of Nanog,a pluripotency sustaining factor in embryonic stem cells[J].Cell,113(5):643-655.
Choulika A,Perrin A,Dujon B,et al.1995.Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae[J].Molecular and Cellular Biology,15(4):1968-1973.
Cong L,Ran F A,Cox D,et al.2013.Multiplex genome engineering using CRISPR/Cas system[J].Science,339(6121):891-823.
Cowan C A,Atienza J,Melton D A,et al.2005.Nuclear reprogramming of somatic cells after fusion with human em bryonic stem cells[J].Science,309(5739):1369-1373.
Evans M J,Kaufman M H.1981.Establishment in culture of pluripotential cells from mouse embryos[J].Nature,292(5819):154-156.
Freshney R I.2000.Culture of animal cells:A manual of basic technique[M].New York:Wiley-Liss,149-175.
Hart A H,Hartley L,Lbrahim M,et al.2004.Identification cloning and expression analysis of the pluripoteney promoting Nanog genes in mouse and human[J].Developmental Dynamics,230(1):187-198.
Jirimutu,Wang Z,Ding G H,et al.2012.Genome sequences of wild and domestic bactrian camels[J].Nature Communications,3:1202.
Liao J,Cui C,Chen S,et al.2009.Generation of induced pluripotent stem cell lines from adult rat cells[J].Cell Stem Cell,4(1):11-15.
Marius W,Alexander M,Ruth F,et al.2007.In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state[J].Nature,448(7151):318-124.
Meissner A,Wernig M,Jaenisch R.2007.Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells[J].Nature Biotechnology,25(10):1177-1181.
Mitsui K,Tokuzaw Y,Itoh H,et al.2003.The home protein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells[J].Cell,113(5):631-642.
Nimet M,Rupa S,Wei X,et al.2007.Directly reprogrammedfibroblasts show global epigenetic remodeling and widespread tissue contribution[J].Cell Stem Cell,1(1):55-70.
Okita K,Ichisaka T,Yamanaka S.2007.Generation of germline-competent induced pluripotent stem cells[J].Nature,448(7151):313-317.
Peters J,Driesch A.1997.The two humped camel(Camelusbactrianus):New light on its distribution management and medical treatment in the past[J].Journal of Zoology,242:651-679.
Plessis A,Perrin A,HaberJ,et al.1992.Site-specific recombination determined by I-Scel,a mitochondrial group I intron-encoded endonuclease expressed in the yeast nucleus[J].Genetics,130(3):451-460.
Rouet P,Smih F,Jasin M.1994.Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease[J].Molecular and Cellular Biology,14(12):8096-8106.
Tada M,Takahama Y,Abe K,et al.2001.Nuclear reprogramming of somatic cells by in vitro hybridization with EScells[J].Current Biology,11(19):1553-1558.
Takahashi K,Yamanaka S.2006.Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[J].Cell,126(4):663-676.
Takahashi K,Tanabe K,Ohnuki M,et al.2007.Induction of pluripotent stem cells from adult human fibroblasts by defined factors[J].Cell,131(5):861-872.
Wilmut I,Schnieke A E,McWhir J,et al.1997.Viable offspring derived from fetal and adult mammalian cells[J].Nature,385(6619):810-813.
Yamagchi S,Kimura H,Tada M,et al.2005.Nanog expression in mouse germ cell development[J].Gene Expression Patterns,5(5):639-646.