利用CRISPR/Cas9系统在Beta-TC-6细胞株中剔除Sidt2基因
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摘要
目的:利用CRISPR/Cas9基因编辑系统和电穿孔技术,在小鼠胰岛素瘤胰岛β细胞(Beta-TC-6)中剔除溶酶体膜蛋白Sidt2基因。方法:针对小鼠Sidt2基因设计向导RNA(sgRNA),sgRNA退火合成双链后克隆到px459载体中。测序鉴定成功的重组质粒命名为px459-Sidt2并大量扩增。使用NEPA 21高效基因转染系统将px459-Sidt2重组质粒电转到Beta-TC-6细胞中,电转染48 h后使用嘌呤霉素加压进行阳性细胞筛选。阳性细胞扩增冻存,得到Sidt2基因剔除混合克隆细胞株,提取细胞DNA及蛋白,利用T7酶以及Western blot方法检测细胞株中Sidt2的敲除效果。结果:成功构建靶向小鼠Sidt2基因的px459重组质粒px45-Sidt2。使用NEPA 21高效基因转染系统电转Beta-TC-6的最佳电转参数为脉冲电压150 V,脉冲时间5 ms,脉冲间隔50 ms,脉冲次数2次,电压衰减10%,电穿孔模式为正方向。与对照组细胞相比,嘌呤霉素加压筛选得到的Beta-TC-6阳性细胞中的Sidt蛋白表达缺失(P<0.05),成功在小鼠胰岛素瘤胰岛β细胞(Beta-TC-6)中剔除溶酶体膜蛋白Sidt2基因。结论:利用CRISPR/Cas9基因编辑系统以及电穿孔技术,在相对难转染的小鼠胰岛素瘤胰岛β细胞Beta-TC-6成功剔除Sidt2基因。
Objective: To knock out the lysosomal membrane protein Sidt2 gene in mouse pancreatic islet tumor cells(Beta-TC-6) using the CRISPR/Cas9 gene editing technique.Methods:Single-guided RNAs(sgRNAs) target on Sidt2 gene was designed,and colonized into BbsI site in px459 after annealing.The correct recombinant plasmids were identified by sequencing,named as px459-Sidt2,and subjected to amplification and sequence verification.Then the recombinant plasmids were extracted with EndoFree Maxi Plasmid Kit to achieve the purity and concentration required for cell electrical transfection.The px459-Sidt2 recombinant plasmid was transfected into Beta-TC-6 cells using NEPA 21 efficient gene transfection system,and the positive cells were screened with the pressure of puromycin for 48 hours following transfection.The positive cells were amplified and frozen to obtain mixed clone cell lines from which Sidt2 gene was deleted.The cell DNA and protein were extracted,and the knockout effect of Sidt2 was detected using T7 enzyme and Western blot.Results:px459 recombinant plasmid targeting mouse Sidt2 gene was successfully constructed.The optimal electrotransport parameter of electroporation Beta-TC-6 using NEPA 21 high-efficiency gene transfection system was at impulse voltage 150 V,time 5 ms,interval 50 ms,frequency 2 times,and voltage attenuation by 10%.Compared with control cells,sidt protein expression in puromycin-screened Beta-TC-6 positive cells was absent.Conclusion:The Sidt2 gene was successfully knocked out in Beta-TC-6,a relatively difficult-to-transfect mouse pancreatic tumor cell line using CRISPR/Cas9 gene editing and electroporation.
Objective: To knock out the lysosomal membrane protein Sidt2 gene in mouse pancreatic islet tumor cells(Beta-TC-6) using the CRISPR/Cas9 gene editing technique.Methods:Single-guided RNAs(sgRNAs) target on Sidt2 gene was designed,and colonized into BbsI site in px459 after annealing.The correct recombinant plasmids were identified by sequencing,named as px459-Sidt2,and subjected to amplification and sequence verification.Then the recombinant plasmids were extracted with EndoFree Maxi Plasmid Kit to achieve the purity and concentration required for cell electrical transfection.The px459-Sidt2 recombinant plasmid was transfected into Beta-TC-6 cells using NEPA 21 efficient gene transfection system,and the positive cells were screened with the pressure of puromycin for 48 hours following transfection.The positive cells were amplified and frozen to obtain mixed clone cell lines from which Sidt2 gene was deleted.The cell DNA and protein were extracted,and the knockout effect of Sidt2 was detected using T7 enzyme and Western blot.Results:px459 recombinant plasmid targeting mouse Sidt2 gene was successfully constructed.The optimal electrotransport parameter of electroporation Beta-TC-6 using NEPA 21 high-efficiency gene transfection system was at impulse voltage 150 V,time 5 ms,interval 50 ms,frequency 2 times,and voltage attenuation by 10%.Compared with control cells,sidt protein expression in puromycin-screened Beta-TC-6 positive cells was absent.Conclusion:The Sidt2 gene was successfully knocked out in Beta-TC-6,a relatively difficult-to-transfect mouse pancreatic tumor cell line using CRISPR/Cas9 gene editing and electroporation.
引文
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[16] TAKEI H,BABA Y,HISATSUNE A,et al.Glycyrrhizin inhibits interleukin-8 production and nuclear factor-kappaB activity in lung epithelial cells,but not through glucocorticoid receptors[J].J Pharmacol Sci,2008,106(3):460-468.
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[18] XU Y,LU Y,XING W.An individually addressable suspended-drop electroporation system for high-throughput cell transfection[J].Lab Chip,2014,14(4):686-690.
[2] MOJICA FJ,DIEZ-VILLASENOR C,GARCIA-MARTINEZ J,et al.Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements[J].J Mol Evol,2005,60(2):174-182.
[3] POURCEL C,SALVIGNOL G,VERGNAUD G.CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA,and provide additional tools for evolutionary studies[J].Microbiology,2005,151(Pt 3):653-663.
[4] DOUDNA JA,CHARPENTIER E.Genome editing.The new frontier of genome engineering with CRISPR-Cas9[J].Science,2014,346(6213):1258096.
[5] WANG H,YANG H,SHIVALILA CS,et al.One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering[J].Cell,2013,153(4):910-918.
[6] YANG H,WANG H,SHIVALILA CHIKDU S,et al.One-step generation of mice carrying reporter and conditional alleles by crispr/cas-mediated genome engineering[J].Cell,2013,154(6):1370-1379.
[7] LIANG X,POTTER J,KUMAR S,et al.Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection[J].Journal of Biotechnology,2015,208:44-53.
[8] CONG L,RAN FA,COX D,et al.Multiplex genome engineering using CRISPR/Cas systems[J].Science,2013,339(6121):819-823.
[9] MILLER JC,TAN S,QIAO G,et al.A TALE nuclease architecture for efficient genome editing[J].Nature Biotechnology,2010,29:143.
[10] ZHOU X,XIN J,FAN N,et al.Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer[J].Cell Mol Life Sci,2015,72(6):1175-1184.
[11] WAN H,FENG C,TENG F,et al.One-step generation of p53 gene biallelic mutant Cynomolgus monkey via the CRISPR/Cas system[J].Cell Res,2015,25(2):258-261.
[12] ZHAO P,ZHANG Z,KE H,et al.Oligonucleotide-based targeted gene editing in C.elegans via the CRISPR/Cas9 system[J].Cell Res,2014,24(2):247-250.
[13] HRUSCHA A,KRAWITZ P,RECHENBERG A,et al.Efficient CRISPR/Cas9 genome editing with low off-target effects in zebrafish[J].Development,2013,140(24):4982-4987.
[14] SHAN Q,WANG Y,LI J,et al.Targeted genome modification of crop plants using a CRISPR-Cas system[J].Nat Biotechnol,2013,31(8):686-688.
[15] MORGENS DW,WAINBERG M,BOYLE EA,et al.Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens[J].Nature Communications,2017,8:15178.
[16] TAKEI H,BABA Y,HISATSUNE A,et al.Glycyrrhizin inhibits interleukin-8 production and nuclear factor-kappaB activity in lung epithelial cells,but not through glucocorticoid receptors[J].J Pharmacol Sci,2008,106(3):460-468.
[17] FAURIE C,REBERSEK M,GOLZIO M,et al.Electro-mediated gene transfer and expression are controlled by the life-time of DNA/membrane complex formation[J].J Gene Med,2010,12(1):117-125.
[18] XU Y,LU Y,XING W.An individually addressable suspended-drop electroporation system for high-throughput cell transfection[J].Lab Chip,2014,14(4):686-690.