Please wait a minute...

中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2014, Vol. 34 Issue (7): 44-48    DOI: 10.13523/j.cb.20140707
技术与方法     
线粒体转录因子A敲低转基因小鼠的研制
秦瑶, 赵鸿彦, 张文航, 王冬梅
遵义医学院附属医院 遵义 563003
Generation of Mitochondrial Transcription Factor a Knockdown Transgenic Mice
QIN Yao, ZHAO Hong-yan, ZHANG Wen-hang, WANG Dong-mei
Affiliated Hospital of Zunyi Medical Colleague, Zunyi 563003, China
 全文: PDF(783 KB)   HTML
摘要:

目的:制备线粒体缺陷的转基因小鼠,为研究线粒体相关疾病提供一个较好的动物模型。方法:将线粒体转录因子A(mitochondrial transcription factor A,TFAM)基因干扰慢病毒显微注射到小鼠受精卵中制备转基因小鼠。将转基因小鼠各组织制备冰冻切片观察荧光表达。用定量PCR(quantitative PCR,qPCR)和Western blot检测心脏TFAM基因的变化。用定量PCR检测心脏线粒体DNA拷贝数的变化。结果:首先构建了针对TFAM基因的干扰慢病毒载体,使用此病毒体外转染细胞后,能够有效的下调TFAM基因的转录和翻译。使用此病毒进行小鼠受精卵卵周注射,可以获得相应的转基因小鼠。病毒携带的绿色荧光蛋白在转基因小鼠的各个组织器官中均能很好的表达。转基因小鼠各组织TFAM转录水平明显降低。此转基因小鼠心脏中TFAM蛋白表达被有效的抑制,心肌细胞线粒体基因组拷贝数下降,心肌细胞线粒体呼吸链功能受损。结论:成功获得了线粒体转录因子A敲低的转基因小鼠。

关键词: 线粒体线粒体转录因子A慢病毒转基因小鼠    
Abstract:

Objective: To produce mitochondria deficiency transgenic mice, providing a useful animal model for the investigation of mitochondria related diseases. Methods: Transgenic mice were produced by injection of mitochondrial transcription factor A (TFAM) RNAi lentivirus into zygotes. Frozen sections of tissues wre prepared to observe GFP expression. Variations of TFAM mRNA and protein were separately detected by quantitative PCR (qPCR) and western blot. Changes of mtDNA copy number were measured by qPCR. Results: We constructed lentiviral vectors expressing shRNA targeting TFAM, which markedly repressed the transcription and translation of TFAM in transduced MEF cells. TFAM knockdown mice were successfully produced by infection of the purified virus into the perivitelline space of single-cell embryo. Green fluorescence was observed in heart, liver, spleen, lung and kidney of transgenic mice. Using qPCR and western blot analysis, we found that the transcription of TFAM in the tissues of transgenic mice was significantly decreased. Moreover, reduced expression of TFAM in myocardium led to a decline of mitochondrial DNA (mtDNA) copy number, as well as an impaired respiratory chain function. Conclusion: A TFAM knockdown transgenic mice line was successfully constructed.

Key words: Mitochondria    Mitochondrial transcription factor A    Lentivirus    Transgenic mice
收稿日期: 2014-04-22 出版日期: 2014-07-25
ZTFLH:  R54  
基金资助:

贵州省省长资金临床应用课题专项研究黔省专合字(2012)180号资助项目

通讯作者: 秦瑶     E-mail: cloudytime@126.com
服务  
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章  

引用本文:

秦瑶, 赵鸿彦, 张文航, 王冬梅. 线粒体转录因子A敲低转基因小鼠的研制[J]. 中国生物工程杂志, 2014, 34(7): 44-48.

QIN Yao, ZHAO Hong-yan, ZHANG Wen-hang, WANG Dong-mei. Generation of Mitochondrial Transcription Factor a Knockdown Transgenic Mice. China Biotechnology, 2014, 34(7): 44-48.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140707        https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I7/44


[1] Jin H,May M,Tranebjaerg L,et al. A novel X-linked gene,DDP,shows mutations in families with deafness(DFN-1),dystonia,mental deficiency and blindness. Nat Genet,1996,14(2): 177-180.

[2] Hudson G,Deschauer M,Busse K,et al. Sensory ataxic neuropathy due to a novel C10Orf2 mutation with probable germline mosaicism.Neurology,2005,64(2): 371-373.

[3] Kemp J P,Smith P M,Pyle A,et al. Nuclear factors involved in mitochondrial translation cause a subgroup of combined respiratory chain deficiency.Brain,2011,134(Pt 1): 183-195.

[4] Lemieux H, Semsroth S, Antretter H, et al. Mitochondrial respiratory control and early defects of oxidative phosphorylation in the failing human heart. Int J Biochem Cell Biol,2011,43(12):1729-1738.

[5] Karamanlidis G, Bautista-Hernandez V, Fynn-Thompson F, et al. Impaired mitochondrial biogenesis precedes heart failure in right ventricular hypertrophy in congenital heart disease. Circ Heart Fail,2011,4(6):707-713.

[6] Lu N, Sun Y, Zheng X. Orientin-induced cardioprotection against reperfusion is associated with attenuation of mitochondrial permeability transition. Planta Med,2011,77(10):984-991.

[7] Shokolenko I, Venediktova N, Bochkareva A, et al. Oxidative stress induces degradation of mitochondrial DNA. Nucleic Acids Res,2009,37(8):2539-2548.

[8] Clayton D A. Replication and transcription of vertebrate mitochondrial DNA. Annu Rev Cell Biol,1991,7:453-478.

[9] Campbell C T, Kolesar J E, Kaufman B A. Mitochondrial transcription factor A regulates mitochondrial transcription initiation, DNA packaging, and genome copy number. Biochim Biophys Acta,2012,1819(9-10):921-929.

[10] Tyynismaa H, Sembongi H, Bokori-Brown M, et al. Twinkle helicase is essential for mtDNA maintenance and regulates mtDNA copy number. Hum Mol Genet,2004,13(24):3219-3227.

[11] Wibom R, Hagenfeldt L, von Döbeln U. Measurement of ATP production and respiratory chain enzyme activities in mitochondria isolated from small muscle biopsy samples. Anal Biochem,2002,311(2):139-51.

[12] Hance N, Ekstrand M I, Trifunovic A. Mitochondrial DNA polymerase gamma is essential for mammalian embryogenesis. Hum Mol Genet,2005,14(13): 1775-1783.

[13] Larsson N G, Wang J, Wilhelmsson H, et al. Mitochondrial transcription factor A is necessary for mtDNA maintenance and embryogenesis in mice. Nat Genet,1998,18(3):231-236.

[14] Tavi P, Hansson A, Zhang S J, et al. Abnormal Ca(2+) release and catecholamine-induced arrhythmias in mitochondrial cardiomyopathy. Hum Mol Genet,2005,14(8): 1069-1076.

[15] Shin K J, Wall E A, Zavzavadjian J R, et al. A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated transgene expression. Proc Natl Acad Sci U S A,2006,103(37):13759-13764.

[16] Stegmeier F, Hu G, Rickles R J, et al. A lentiviral microRNA-based system for single-copy polymerase II-regulated RNA interference in mammalian cells. Proc Natl Acad Sci U S A,2005,102(37):13212-13217.

[17] Lois C, Hong E J, Pease S, et al. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science,2002,295(5556):868-872.

[1] 赵晓煜,徐祺玲,赵晓东,安云飞. 基因治疗慢病毒载体的转导增强策略*[J]. 中国生物工程杂志, 2021, 41(8): 52-58.
[2] 钱昱,丁晓雨,刘志强,袁增强. 基因修饰人多能干细胞的高效单克隆建系方法[J]. 中国生物工程杂志, 2021, 41(8): 33-41.
[3] 段阳阳,张凤亭,成江,石瑾,杨娟,李海宁. SIRT2抑制对MPP+诱导的帕金森病细胞模型凋亡和线粒体动态平衡的影响*[J]. 中国生物工程杂志, 2021, 41(4): 1-8.
[4] 蔡润泽,王正波,陈永昌. Mecp2影响Rett综合征中代谢功能的研究进展 *[J]. 中国生物工程杂志, 2021, 41(2/3): 89-97.
[5] 张晨阳,黑常春,袁仕林,周玉佳,曹美玲,秦亦欣,杨笑. SIRT3抑制线粒体自噬并减轻高糖加重的神经元缺氧再灌注损伤*[J]. 中国生物工程杂志, 2021, 41(11): 1-13.
[6] 黄胜, 严启滔, 熊仕琳, 彭弈骐, 赵蕊. 基于CRISPR/Cas9-SAM系统CHD5基因过表达慢病毒载体的构建及对膀胱癌T24细胞增殖,迁移和侵袭能力的影响 *[J]. 中国生物工程杂志, 2020, 40(3): 1-8.
[7] 梁振鑫,刘芳,张玮,刘庆友,李力. 抗p185 erb B2人鼠嵌合抗体ChAb26转基因小鼠乳腺生物反应器的制备与验证 *[J]. 中国生物工程杂志, 2019, 39(8): 40-51.
[8] 菅璐,黄映辉,梁天亚,王利敏,马洪涛,张婷,李丹阳,王明连. 利用CRISPR/Cas9技术建立敲除JAK2基因K562细胞系 *[J]. 中国生物工程杂志, 2019, 39(7): 39-47.
[9] 马占兵,党洁,杨继辉,霍正浩,徐广贤. 基于慢病毒系统的双荧光标记多功能自噬流监测系统建立与应用 *[J]. 中国生物工程杂志, 2019, 39(5): 88-95.
[10] 李凤,高晓冬,中西秀树. 在酿酒酵母中研究人类mOGT基质导向序列的功能 *[J]. 中国生物工程杂志, 2019, 39(4): 32-37.
[11] 韩亚丽,杨冠恒,陈雁雯,龚秀丽,张敬之. 表达β-珠蛋白基因的安全性慢病毒载体的优化 *[J]. 中国生物工程杂志, 2018, 38(7): 50-57.
[12] 韩明明,罗玉萍. 内源CD133 +细胞示踪小鼠模型的制备和鉴定 *[J]. 中国生物工程杂志, 2018, 38(6): 58-62.
[13] 刘艳艳,李会荣,胡悦,范阳阳,李祥明,谭晴晴,吴家强,步迅. 饲料中狐狸、水貂、貉子和狗源性的五重实时荧光PCR检测方法的建立 *[J]. 中国生物工程杂志, 2017, 37(12): 67-76.
[14] 葛林, 刘新宇, WANG Guirong. 人SP-B蛋白转基因小鼠及细菌性肺炎模型的构建[J]. 中国生物工程杂志, 2017, 37(10): 65-71.
[15] 左志宇, 辛灵彪, 杨洁, 王鑫廷. SND1转基因小鼠的构建[J]. 中国生物工程杂志, 2016, 36(4): 97-103.