腺病毒载体转染水貂耳缘成纤维细胞的研究

doi:10.16431/j.cnki.1671-7236.2020.01.005

摘要/Abstract

摘要： 试验旨在探究利用携带标记基因增强型绿色荧光蛋白（enhanced green fluorescent protein，EGFP）的腺病毒载体转染入水貂耳缘成纤维细胞的可行性及感染效率。首先利用组织贴壁法分离培养水貂原代耳缘成纤维细胞并进行传代，然后包装病毒Ad-EGFP并使用TCID₅₀法进行滴度检测，设置两组不同浓度梯度的病毒液感染水貂耳缘成纤维细胞，感染复数（MOI）值分别为0/100/300/500/700/900和0/10/30/50/70/90，通过在显微镜下观察阳性细胞占总细胞数的比值估计转染效率（%）。结果表明，组织贴壁法培养水貂耳皮肤组织11 d可长满原代细胞，传代细胞在3~4 d融合度可达80%左右，包装Ad-EGFP病毒滴度为1.99×10¹¹ PFU/mL，用其感染水貂成纤维细胞最佳MOI值为30，瞬时转染效率可达90%以上。综上，利用腺病毒载体转染水貂成纤维细胞具有可行性，是一种高效的转染方式，为后续水貂基因组编辑前期验证试验奠定基础。

关键词: 水貂; 腺病毒转染; 成纤维细胞; 细胞培养

Abstract: The objective of this study was to investigate the feasibility and efficiency of transfection of adenovirus vector with enhanced green fluorescent protein (EGFP) into fibroblasts in the ear margin of mink.Firstly,ear fibroblasts of mink were isolated and cultured by tissue adherent method,and then the virus Ad-EGFP was packaged and the titer was detected by TCID₅₀ method.Two groups of different concentration gradients were used to infect mink fibroblasts,multiplicity of infection(MOI):0/100/300/500/700/900 and 0/10/30/50/70/90.Transfection efficiency (%) was estimated by looking at the ratio of positive cells to the total number of cells under a microscope.The results showed that primary cells could be grown in the ear tissue of mink cultured by tissue adherent method for 11 d,and the fusion degree of passage cells could reach 80% in 3-4 d.The titer of the packaged Ad-EGFP virus was 1.99×10¹¹ PFU/mL,the optimal MOI value of the infected mink fibroblasts was 30,and the instantaneous transfection efficiency was more than 90%.In conclusion,it was feasible to transfection mink fibroblasts with adenovirus vectors,which was an efficient transfection method and provided a basis for the preliminary validation test of mink genome editing.

Key words: American mink; adenovirus transfection; fibroblast; cell culture

中图分类号:

S855.3

李文, 韩玉萍, 赵向远, 范冰峰, 李晓霞, 刁云飞, 杨镒峰, 许保增. 腺病毒载体转染水貂耳缘成纤维细胞的研究[J]. 中国畜牧兽医, 2020, 47(1): 37-43.

LI Wen, HAN Yuping, ZHAO Xiangyuan, FAN Bingfeng, LI Xiaoxia, DIAO Yunfei, YANG Yifeng, XU Baozeng. American Mink Ear Fibroblasts Transfected by Adenovirus Vector[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(1): 37-43.

参考文献

[1] KARIMI K,SARGOLZAEI M,WANG Z.Genetic trends for reproductive traits in American mink[J].Journal of Animal Science,2018,96(7):115-116.
[2] EZHKOV V O,EZHKOVA M S,YAPPAROV I A.Ultrastructure and nanomorphology of the American mink (Mustela vison) kidney[J].Doklady Biological Sciences,2019,485(1):56-58.
[3] JINEK M,CHYLINSKI K,FONFARA I,et al.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J].Science,2012,337(6096):816-821.
[4] SAYEDAHMED E E,KUMARI R,MITTAL S K.Current use of adenovirus vectors and their production methods[J].Methods in Molecular Biology,2019,1937:155-175.
[5] ZHU X X,ZHONG Y Z,GE Y W.Generation of transgenic-cloned Huanjiang Xiang pigs systemically expressing enhanced green fluorescent protein[J].Reproduction in Domestic Animals,2018,53(6):1546-1554.
[6] LI L F,GUAN W J,HUA Y,et al.Establishment and characterization of a fibroblast cell line from the Mongolian horse[J].In Vitro Cellular & Developmental Biology Animal,2009,45(7):311-316.
[7] 曹素芳.靶向HP-PRRSV ORF7基因siRNA体外抑制HP-PRRSV HN1株复制研究[J].中国兽医杂志,2018,54(6):25-28. CAO S F.Inhibition of replication of HP-PRRSV HN1 strain in vitro by siRNA targeting the ORF7 gene of HP-PRRSV[J].Chinese Journal of Veterinary Medicine,2018,54(6):25-28.(in Chinese)
[8] 张大鹏,李跃民,栗楠,等.广西巴马小型猪皮肤成纤维细胞的分离培养体系优化[J].生物学杂志,2017,34(2):99-103. ZHANG D P,LI Y M,LI N,et al.Isolation and culture of skin fibroblasts in Guangxi Bama mini-pig[J].Journal of Biology,2017,34(2):99-103.(in Chinese)
[9] 邱桂斌,李祥瑞.德国牧羊犬成纤维细胞培养及其生长特性研究[J].中国畜牧兽医,2012,39(4):119-121. QIU G B,LI X R.The Culture and growth characteristics studies on canine fibroblast cell[J].China Animal Husbandry & Veterinary Medicine,2012,39(4):119-121.(in Chinese)
[10] 张旗,郭晓红,高鹏飞,等.马身猪耳缘组织成纤维细胞的体外培养与鉴定[J].中国畜牧兽医,2017,44(8):2289-2294. ZHANG Q,GUO X H,GAO P F,et al.In vitro culture and identification of ear marginal fibroblast cell lines derived from Mashen pigs[J].China Animal Husbandry & Veterinary Medicine,2017,44(8):2289-2294.(in Chinese)
[11] CERRATO S,RAMIÓ-LLUCH L,BRAZÍS P,et al.Development and characterization of an equine skin-equivalent model[J].Veterinary Dermatology,2014,25(5):475-477.
[12] TOMASELLO L,MUSSO R.Donor age and long- term culture do not negatively influence the stem potential of limbal fibroblast- like stem cells[J].Stem Cell Research & Therapy,2016,71(8):77-83.
[13] GAO S,ZHENG C,CHANG G,et al.Unique features of mutations revealed by sequentially reprogrammed induced pluripotent stem cells[J].Nature Communications,2015,12(6):6318-6326.
[14] 范晓梅,张通,栗瑞兰,等.绒山羊附睾上皮细胞培养方法的建立及其生长特性[J].畜牧兽医学报,2017,48(7):1212-1220. FAN X M,ZHANG T,LI R L,et al.Establishment of culture method and growth characteristics for epididymal epithelial cells from cashmere goats[J].Acta Veterinaria et Zootechnica Sinica,2017,48(7):1212-1220.(in Chinese)
[15] 崔小荣,李晓玲,于光辉,等.转pGH基因猪与非转基因猪成纤维细胞系的建立及鉴定[J].中国兽医杂志,2017,53(6):3-7. CUI X R,LI X L,YU G H,et al.The establishment and identification of fibroblast cell lines with pGH transgenic pigs andnon-transgenic pigs[J].Chinese Journal of Animal Science,2017,53(6):3-7.(in Chinese)
[16] CROOKS D R,FAN T W,LINEHAN W M.Metabolic labeling of cultured mammalian cells for stable isotope-resolved metabolomics:Practical aspects of tissue culture and sample extraction[J].Methods in Molecular Biology,2019,1928:1-27.
[17] SARAH E,KWAN,JORDAN P,et al.Comparing bacterial,fungal,and human cell concentrations with rapid adenosine triphosphate measurements for indicating microbial surface contamination[J].American Journal of Infection Control,2019,47(6):671-676.
[18] LIN J,CHEN L,JIANG W,et al.Rapid detection of low-level HeLa cell contamination in cell culture using nested PCR[J].Journal of Cellular and Molecular Medicine,2019,23(1):227-263.
[19] ARENA T A,HARMS P D,WONG A W.High throughput transfection of HEK293 cells for transient protein production[J].Methods in Molecular Biology,2018,1850:179-187.
[20] 李希,王曦,吴苏君.吕梁黑山羊耳缘成纤维细胞的分离及体外培养[J].山西农业科学,2018,46(7):1186-1189. LI X,WANG X,WU S J.Separation and in vitro culture of the Lüliang Black goat ear fibroblasts[J].Journal of Shanxi Agricultural Sciences,2018,46(7):1186-1189.(in Chinese)
[21] 李虎,李儒军,曹宸喜,等.强直性脊柱炎髋关节韧带组织培养模型的建立及腺病毒转染的实验研究[J].中华骨与关节外科杂志,2018,11(12):932-939. LI H,LI R J,CAO C X,et al.Establishment of culture models for hip joint tendons of ankylosing spondylitis and the effect of adenovirus-mediated gene transfer[J].Chinese Journal of Bone and Joint Surgery,2018,11(12):932-939.(in Chinese)
[22] RYBAKOVSKY E,VALENZANO M C,DIGUILIO K M,et al.Improving transient transfection efficiency in a differentiated,polar epithelial cell layer[J].Journal of Biomolecular Techniques,2019,30(2):19-24.
[23] KORNBERG L J,GRANT M B.Adenoviruses increase endothelial cell proliferation,migration,and tube formation:Partial reversal by the focal adhesion kinase inhibitor,FRNK[J].Microvascular Research,2007,73(3):157-162.
[24] PALMER D J,TURNER D L,NG P.Production of CRISPR/Cas9-mediated self-cleaving helper-dependent adenoviruses[J].Molecular Therapy Methods & Clinical Development,2019,16(13):432-439.