脂质体介导基因编辑载体质粒转染条件的优化
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摘要
[目的]探讨脂质体介导基因编辑载体质粒转染人食管癌Eca-109细胞的最佳条件。[方法]以基于Cre/Lox P系统的p GT-A1B1和基于CRISPR/Cas9系统的p X458-sgRNA8两种基因编辑载体质粒为实验材料,将环状和线性质粒以不同DNA用量(0. 2、0. 4、0. 6和0. 8μg)和不同DNA与脂质体比例(1∶1、1∶1. 5、1∶2、1∶2. 5和1∶3)转染Eca-109细胞,计算转染效率。[结果]不同DNA用量条件下,p GT-A1B1在0. 4μg和0. 6μg时转染效率较高,p X458-sgRNA8在0. 6μg时转染效率较高,与其它DNA用量比较均有显著性差异(P <0. 05)。不同DNA与脂质体比例条件下,p GT-A1B1在比例为1∶1. 5和1∶2时转染效率较高,p X458-sgRNA8在比例为1∶2. 5时转染效率较高,与其它比例比较均有显著性差异(P <0. 05)。同一种质粒的环状和线性结构形态,在DNA用量或DNA与脂质体比例相同时,转染效率差异不显著(P> 0. 05)。[结论]建立了脂质体介导基因编辑载体质粒转染Eca-109细胞的最佳转染条件,质粒p GT-A1B1和p X458-sgRNA8的DNA用量分别为0. 4～0. 6μg和0. 6μg,DNA与脂质体比例分别为1∶1. 5～1∶2和1∶2. 5。
[Objective] To investigate the optimal conditions for transfection of liposome mediated gene editing vector plasmid into human esophageal cancer Eca-109 cells. [Method]Using p GT-A1 B1 based on Cre/Lox P system and p X458-sgRNA8 based on CRISPR/Cas9 system as experimental materials,circular and linear plasmids were transfected into Eca-109 cells with different DNA amounts( 0. 2,0. 4,0. 6 and 0. 8 μg) and different DNA-liposome ratios( 1 ∶ 1,1 ∶ 1. 5,1 ∶ 2,1 ∶ 2. 5 and1∶3),and the transfection efficiency was calculated.[Result]Under different DNA amount conditions,the transfection efficiency of p GT-A1 B1 was higher at 0. 4 and 0. 6 μg,and that of p X458-sgRNA8 was higher at 0. 6 μg,which had significant difference compared with other DNA amounts( P < 0. 05). Under different ratios of DNA to liposome,the transfection efficiency of p GT-A1 B1 was higher when the ratio was 1∶ 1. 5 and 1∶ 2,while that of p X458-sgRNA8 was higher when the ratio was1∶2. 5,which had significant difference compared with other ratios( P < 0. 05). There was no significant difference in transfection efficiency between the circular and linear structure of the same plasmid when the DNA amount or the ratio of DNA to liposome was the same( P > 0. 05).[Conclusion]The optimal conditions for transfection of Eca-109 cells with liposome mediated gene editing vector plasmid were established. The DNA amounts was 0. 4-0. 6 μg and 0. 6 μg,and the ratios of DNA to liposome was 1∶1. 5-1∶2 and 1∶2. 5,for plasmid p GT-A1 B1 and p X458-sgRNA8 respectively.
[Objective] To investigate the optimal conditions for transfection of liposome mediated gene editing vector plasmid into human esophageal cancer Eca-109 cells. [Method]Using p GT-A1 B1 based on Cre/Lox P system and p X458-sgRNA8 based on CRISPR/Cas9 system as experimental materials,circular and linear plasmids were transfected into Eca-109 cells with different DNA amounts( 0. 2,0. 4,0. 6 and 0. 8 μg) and different DNA-liposome ratios( 1 ∶ 1,1 ∶ 1. 5,1 ∶ 2,1 ∶ 2. 5 and1∶3),and the transfection efficiency was calculated.[Result]Under different DNA amount conditions,the transfection efficiency of p GT-A1 B1 was higher at 0. 4 and 0. 6 μg,and that of p X458-sgRNA8 was higher at 0. 6 μg,which had significant difference compared with other DNA amounts( P < 0. 05). Under different ratios of DNA to liposome,the transfection efficiency of p GT-A1 B1 was higher when the ratio was 1∶ 1. 5 and 1∶ 2,while that of p X458-sgRNA8 was higher when the ratio was1∶2. 5,which had significant difference compared with other ratios( P < 0. 05). There was no significant difference in transfection efficiency between the circular and linear structure of the same plasmid when the DNA amount or the ratio of DNA to liposome was the same( P > 0. 05).[Conclusion]The optimal conditions for transfection of Eca-109 cells with liposome mediated gene editing vector plasmid were established. The DNA amounts was 0. 4-0. 6 μg and 0. 6 μg,and the ratios of DNA to liposome was 1∶1. 5-1∶2 and 1∶2. 5,for plasmid p GT-A1 B1 and p X458-sgRNA8 respectively.
引文
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[13]刘甦苏,吴曦,周舒雅,等.基于Cre-Lox P系统的条件性定点敲入人源hRas基因小鼠的建立[J].实验动物科学,2018,35(4):21-28.
[14]Singh V,Gohil N,Ramírez García R,et al.Recent advances in CRISPR-Cas9 genome editing technology for biological and biomedical investigations[J].J Cell Biochem,2018,119(1):81-94.
[15]郭晓龙,张青峰,野庆松,等.靶向ezrin增强子关键区的CRISPR/Cas9载体的构建[J].生物学杂志,2017,34(6):19-22.
[16]占米林,阚宝军,张辉,等.谷氨酸棒状杆菌CRISPR-Cpf1和Cre/lox P基因敲除技术的比较[J].微生物学通报,2019,46(2):278-291.
[17]黄瑜烨,李碧舟,何颖,等.基于CRISPR/Cas9技术的Ha CaT的TLR4基因定向敲除及其功能初步研究[J].生物技术,2019,29(1):57-62,102.
[18]高岑,史书龙,张旭,等.脂质体介导法转染m IMCD-3细胞的可行性及最佳转染条件[J].中国组织工程研究与临床康复,2011,15(15):2739-2742.
[19]廖维芳,李建玲,孙芳,等.优化脂质体对肝癌细胞株HepG2细胞转染效率[J].基因组学与应用生物学,2016,35(9):2229-2234.
[20]赵轶男,张树彪,崔韶晖,等.阳离子脂质体介导基因转染肿瘤细胞[J].中国细胞生物学学报,2013,35(10):1459-1464.
[2]White MK,Kaminski R,Young WB,et al.CRISPR editing technology in biological and biomedical investigation[J].J Cell Biochem,2017,118(11):3586-3594.
[3]Nguyen TH,Anegon I.Successful correction of hemophilia by CRISPR/Cas9 genome editing in vivo:delivery vector and immune responses are the key to success[J].EMBO Mol Med,2016,8(5):439-441.
[4]Wong TWY,Cohn RD.Therapeutic applications of CRISPR/Cas for duchenne muscular dystrophy[J].Curr Gene Ther,2017,17(4):301-308.
[5]Cavazzana M,Mavilio F.Gene therapy for hemoglobinopathies[J].Hum Gene Ther,2018,29(10):1106-1113.
[6]Yang S,Chang R,Yang H,et al.CRISPR/Cas9-mediated gene editing ameliorates neurotoxicity in mouse model of Huntington's disease[J].J Clin Invest,2017,127(7):2719-2724.
[7]Schenborn ET,Oler J.Liposome-mediated transfection of mammalian cells[J].Methods Mol Biol,2000,130:155-164.
[8]Levine AJ.Virus vector-mediated gene transfer[J].Microbiol Sci,1987,4(8):245-250.
[9]Kim H,Kim M,Im SK,et al.Mouse Cre-Lox P system:general principles to determine tissue-specific roles of target genes[J].Lab Anim Res,2018,34(4):147-159.
[10]Ran FA,Hsu PD,Wright J,et al.Genome engineering using the CRISPR-Cas9 system[J].Nat Protoc,2013,8(11):2281-2308.
[11]李军华,韩翠芹,邓捷,等.双筛选标记打靶载体的构建及其功能鉴定[J].生物工程学报,2010,26(12):1696-1703.
[12]王涛,童武,叶超,等.应用Cre/loxp系统构建含有BAC序列的重组伪狂犬病毒[J].中国动物传染病学报,2017,25(2):22-28.
[13]刘甦苏,吴曦,周舒雅,等.基于Cre-Lox P系统的条件性定点敲入人源hRas基因小鼠的建立[J].实验动物科学,2018,35(4):21-28.
[14]Singh V,Gohil N,Ramírez García R,et al.Recent advances in CRISPR-Cas9 genome editing technology for biological and biomedical investigations[J].J Cell Biochem,2018,119(1):81-94.
[15]郭晓龙,张青峰,野庆松,等.靶向ezrin增强子关键区的CRISPR/Cas9载体的构建[J].生物学杂志,2017,34(6):19-22.
[16]占米林,阚宝军,张辉,等.谷氨酸棒状杆菌CRISPR-Cpf1和Cre/lox P基因敲除技术的比较[J].微生物学通报,2019,46(2):278-291.
[17]黄瑜烨,李碧舟,何颖,等.基于CRISPR/Cas9技术的Ha CaT的TLR4基因定向敲除及其功能初步研究[J].生物技术,2019,29(1):57-62,102.
[18]高岑,史书龙,张旭,等.脂质体介导法转染m IMCD-3细胞的可行性及最佳转染条件[J].中国组织工程研究与临床康复,2011,15(15):2739-2742.
[19]廖维芳,李建玲,孙芳,等.优化脂质体对肝癌细胞株HepG2细胞转染效率[J].基因组学与应用生物学,2016,35(9):2229-2234.
[20]赵轶男,张树彪,崔韶晖,等.阳离子脂质体介导基因转染肿瘤细胞[J].中国细胞生物学学报,2013,35(10):1459-1464.