3种转染试剂对牛原代骨骼肌卫星细胞转染条件的优化
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摘要
为了选择适合牛原代骨骼肌卫星细胞的转染试剂与条件,本研究以带有绿色荧光蛋白(EGFP)的质粒pcDNA3.1-EGFP和pEGFP-C1作为外源基因,分别用Lipofectamine 3000、X-tremeGENE HP和FuGENE HD转染牛原代骨骼肌卫星细胞,通过荧光显微镜观察、实时荧光定量PCR及Hoechst 33342染色评价转染效率。结果表明,对于同一时期、同一转染试剂,pcDNA3.1-EGFP转染效果优于pEGFP-C1;不同转染试剂之间,Lipofectamine 3000和X-tremeGENE HP转染效果优于FuGENE HD。Lipofectamine 3000转染后分化72h肌管最明显,FuGENE HD次之,X-tremeGENE HP转染后细胞死亡较多。采用1.0μL Lipofectamine 3000、1.5μL Lipofectamine 3000和X-tremeGENE HP转染细胞后EGFP表达量极显著高于FuGENE HD (P<0.01),但这三者之间差异不显著(P>0.05)。细胞转染效率与EGFP定量结果基本一致,1.0、1.5μL Lipofectamine 3000转染效率较高,分别达26.07%和24.77%,极显著高于FuGENE HD(5.59%)和X-tremeGENE HP(10.87%)的转染效率(P<0.01),但二者之间无显著差异(P>0.05);X-tremeGENE HP的转染效率极显著高于FuGENE HD (P<0.01)。综合考虑转染试剂的用量、细胞毒性及经济效益,本试验初步认为牛原代骨骼肌卫星细胞转染宜采用Lipofectamine 3000,其与质粒DNA的比例为1∶1时转染效率最高。本试验结果可为原代细胞的转染提供重要的参考价值。
To select a suitable transfection reagent and condition for primary bovine skeleltal muscle satellite cells,the pcDNA3.1-EGFP and pEGFP-C1 plasmids with green fluorescent protein(EGFP)were used as exogenous gene to transfect primary bovine skeletal muscle satellite cells by Lipofectamine 3000,X-tremeGENE HP and FuGENE HD in this study.Then the transfection efficiency was evaluated by fluorescence microscopy,Real-time quantitative PCR and Hoechst 33342 staining.The results showed that transfection efficiency of pcDNA3.1-EGFP was better than pEGFP-C1 for the same period and transfection reagent.Among different transfection reagents,the effect of transfections in Lipofectamine 3000 and X-tremeGENE HP were superior to FuGENE HD.Myotubes were most obvious with Lipofectamine 3000 transfection after differentiation 72 h,followed by FuGENE HD,X-tremeGENE HP transfection leaded to more cell death.The expression of EGFP was extremely significantly higher than FuGENE HD after transfection with 1.0μL Lipofectamine 3000,1.5μL Lipofectamine 3000 and X-tremeGENE HP(P<0.01),but there was no significant difference in three groups(P>0.05).The cell transfection efficiency was basically consistent with the result of EGFP quantification,the transfection efficiency of 1.0and 1.5μL Lipofectamine 3000 was extremely significantly higher than FuGENE HD(5.59%)and X-tremeGENE HP(10.87%)(P<0.01),respectively up to 26.07% and 24.77% with no significant difference(P>0.05).The transfection efficiency of X-tremeGENE HP was also extremely significantly higher than FuGENE HD(P<0.01).Comprehensive consideration the amount of transfection reagents,cytotoxicity and economic benefits,this study preliminarily assumed that the transfection of primary bovine skeletal muscle satellite cells should adopt Lipofectamine 3000 transfection reagent,and the transfection efficiency was the highest when the ratio of Lipofectamine 3000 to plasmid DNA was 1∶1.This experiment could provide important references for transfection of primary cells myogenic.
To select a suitable transfection reagent and condition for primary bovine skeleltal muscle satellite cells,the pcDNA3.1-EGFP and pEGFP-C1 plasmids with green fluorescent protein(EGFP)were used as exogenous gene to transfect primary bovine skeletal muscle satellite cells by Lipofectamine 3000,X-tremeGENE HP and FuGENE HD in this study.Then the transfection efficiency was evaluated by fluorescence microscopy,Real-time quantitative PCR and Hoechst 33342 staining.The results showed that transfection efficiency of pcDNA3.1-EGFP was better than pEGFP-C1 for the same period and transfection reagent.Among different transfection reagents,the effect of transfections in Lipofectamine 3000 and X-tremeGENE HP were superior to FuGENE HD.Myotubes were most obvious with Lipofectamine 3000 transfection after differentiation 72 h,followed by FuGENE HD,X-tremeGENE HP transfection leaded to more cell death.The expression of EGFP was extremely significantly higher than FuGENE HD after transfection with 1.0μL Lipofectamine 3000,1.5μL Lipofectamine 3000 and X-tremeGENE HP(P<0.01),but there was no significant difference in three groups(P>0.05).The cell transfection efficiency was basically consistent with the result of EGFP quantification,the transfection efficiency of 1.0and 1.5μL Lipofectamine 3000 was extremely significantly higher than FuGENE HD(5.59%)and X-tremeGENE HP(10.87%)(P<0.01),respectively up to 26.07% and 24.77% with no significant difference(P>0.05).The transfection efficiency of X-tremeGENE HP was also extremely significantly higher than FuGENE HD(P<0.01).Comprehensive consideration the amount of transfection reagents,cytotoxicity and economic benefits,this study preliminarily assumed that the transfection of primary bovine skeletal muscle satellite cells should adopt Lipofectamine 3000 transfection reagent,and the transfection efficiency was the highest when the ratio of Lipofectamine 3000 to plasmid DNA was 1∶1.This experiment could provide important references for transfection of primary cells myogenic.
引文
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[18]DA C M,SIMES S,DE LIMA M C.Improving lipoplex-mediated gene transfer into C6glioma cells and primary neurons[J].Experimental Neurology,2004,187(1):65-75.
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[20]倪银芸,丁雨,吴思思.四种转染试剂对H9C2细胞转染效率的比较[J].华西医学,2017,7:1024-1028.NI Y Y,DING Y,WU S S.Comparison of four different transfection reagents for transfection efficiency of H9C2cells[J].West China Medical Journal,2017,7:1024-1028.(in Chinese)
[21]CARVALHO T G D,PELLENZ F M,LAUREANO A,et al.A simple protocol for transfecting human mesenchymal stem cells[J].Biotechnology Letters,2018,4:1-6.
[22]粟文俊,蒋泓,唐冬生,等.脂质体转染巴马猪胎儿成纤维细胞条件的优化[J].中国农学通报,2011,27(7):300-303.SU W J,JIANG H,TANG D S,et al.Optimization of parameters of exogene transfection of Luchuan pig fetal fibroblasts in vitro mediated by liposome[J].Chinese Agricultural Science Bulletin,2011,27(7):300-303.(in Chinese)
[23]DASS C R.Lipoplex-mediated delivery of nucleic acids:Factors affecting in vivo transfection[J].Journal of Molecular Medicine,2004,82(9):579-591.
[24]ISHIGURO K,WATANABE O,NAKAMURA M,et al.Combinational use of lipid-based reagents for efficient transfection of primary fibroblasts and hepatoblasts[J].Biotechniques,2017,63(1):37-39.
[2]JORDAN E T,COLLINS M,TEREFE J,et al.Optimizing electroporation conditions in primary and other difficult-to-transfect cells[J].Journal of biomolecular techniques,2008,19(5):328-334.
[3]YANG S H,AGCA Y,CHENG P H,et al.Enhanced transgenesis by intracytoplasmic injection of envelopefree lentivirus[J].Genesis,2007,45(4):177-183.
[4]QIAN W,SHI J,YIN X,et al.PP2Aregulates tau phosphorylation directly and also indirectly viaactivating GSK-3β[J].Journal of Alzheimer’s Disease,2010,19(4):1221-1229.
[5]李华玲,周晓霞.阳离子脂质体转染人类骨骼肌原代细胞的初步研究[J].生物学杂志,2006,23(6):8-10.LI H L,ZHOU X X.Study on the transfection o fprimary human skeletal muscle cells with different cationic liposome[J].Journal of Biology,2006,23(6):8-10.(in Chinese)
[6]SINGER O,VERMA I M.Applications of lentiviral vectors for shRNA delivery and transgenesis[J].Current Gene Therapy,2008,8(6):483-488.
[7]CAMPEAU P,CHAPDELAINE P,SEIGNEURIN-VENIN S,et al.Transfection of large plasmids in primary human myoblasts[J].Gene Therapy,2001,8(18):1387-1394.
[8]郑红云,李艳.大鼠胚胎原代海马神经元两种转染方法的比较[J].神经解剖学杂志,2011,27(6):689-692.ZHENG H Y,LI Y.Comparison of two transfected methods for primary hippocampal neurons of embryonic rats[J].Chinese Journal of Neuroanatomy,2011,27(6):689-692.(in Chinese)
[9]LV H,ZHANG S,WANG B,et al.Toxicity of cationic lipids and cationic polymers in gene delivery[J].Journal of Controlled Release,2006,114(1):100-109.
[10]PRYOR P R.Analyzing lysosomes in live cells[J].Methods Enzymol,2012,505:145-157.
[11]KIEFER K,CLEMENT J,GARIDEL P,et al.Transfection efficiency and cytotoxicity of nonviral gene transfer reagents in human smooth muscle and endothelial cells[J].Pharmaceutical Research,2004,21(6):1009-1017.
[12]LIU J,JONES K L,SUMER H,et al.Stable transgene expression in human embryonic stem cells after simple chemical transfection[J].Molecular Reproduction&Development,2009,76(6):580-586.
[13]张文智,李亚,王鹏雁,等.三种转染试剂对PEF细胞与BHK细胞转染效率的比较[J].新疆农业科学,2013,50(4):753-758.ZHNAG W Z,LI Y,WANG P Y,et al.Transfection efficiency of three transfection reagents to PEF cells and BHK cells[J].Xinjiang Agricultural Sciences,2013,50(4):753-758.(in Chinese)
[14]朱玲,陈强,龚金秋,等.不同转染试剂对大鼠原代间质细胞转染效率的比较分析[J].中国畜牧兽医文摘,2016,32(7):48-49.ZHU L,CHEN Q,GONG J Q,et al.Comparison of transfection efficiency of rat primary mesenchymal cells with different transfection reagents[J].Chinese Abstracts of Animal Husbandry and Veterinary Medicine,2016,32(7):48-49.(in Chinese)
[15]LIU H,REN C,ZHU B,et al.High-efficient transfection of human embryonic stem cells by single-cell plating and starvation[J].Stem Cells&Development,2016,25(6):477-491.
[16]吴婷,吴江,金书欣,等.阳离子脂质体Lipofectamine 3000对贴壁细胞、悬浮细胞和原代细胞转染效率比较的研究[J].现代免疫学,2018,2:89-94.WU T,WU J,JIN S X,et al.The study on the transfection efficiency of cationic liposome lipofectamine3000in adhesive cells,suspension cells and primary cells[J].Current Immunology,2018,2:89-94.(in Chinese)
[17]曹洋,尤双,姚洋,等.环状RNA mmu-circPax3.1表达载体的构建及初步验证[J].中国畜牧兽医,2017,45(10):2865-2870.CAO Y,YOU S,YAO Y,et al.Construction and preliminary verification of expression vector of circular RNA mmu-circ-Pax3.1[J].China Animal Husbandry&Veterinary Medicine,2017,44(10):2865-2870.(in Chinese)
[18]DA C M,SIMES S,DE LIMA M C.Improving lipoplex-mediated gene transfer into C6glioma cells and primary neurons[J].Experimental Neurology,2004,187(1):65-75.
[19]LE B O,CHVRE R,MORNET S,et al.Probing the in vitro mechanism of action of cationic lipid/DNA lipoplexes at a nanometric scale[J].Nucleic Acids Research,2011,39(4):1595-1609.
[20]倪银芸,丁雨,吴思思.四种转染试剂对H9C2细胞转染效率的比较[J].华西医学,2017,7:1024-1028.NI Y Y,DING Y,WU S S.Comparison of four different transfection reagents for transfection efficiency of H9C2cells[J].West China Medical Journal,2017,7:1024-1028.(in Chinese)
[21]CARVALHO T G D,PELLENZ F M,LAUREANO A,et al.A simple protocol for transfecting human mesenchymal stem cells[J].Biotechnology Letters,2018,4:1-6.
[22]粟文俊,蒋泓,唐冬生,等.脂质体转染巴马猪胎儿成纤维细胞条件的优化[J].中国农学通报,2011,27(7):300-303.SU W J,JIANG H,TANG D S,et al.Optimization of parameters of exogene transfection of Luchuan pig fetal fibroblasts in vitro mediated by liposome[J].Chinese Agricultural Science Bulletin,2011,27(7):300-303.(in Chinese)
[23]DASS C R.Lipoplex-mediated delivery of nucleic acids:Factors affecting in vivo transfection[J].Journal of Molecular Medicine,2004,82(9):579-591.
[24]ISHIGURO K,WATANABE O,NAKAMURA M,et al.Combinational use of lipid-based reagents for efficient transfection of primary fibroblasts and hepatoblasts[J].Biotechniques,2017,63(1):37-39.