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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2018, Vol. 38 Issue (9): 55-58    DOI: 10.13523/j.cb.20180908
技术与方法     
地高辛标记Ucp2基因RNA探针的制备和应用 *
刘志贞1,加三三1,2,张凯丽1,孙雨晴1,赵虹1,郝宇卉3,牛勃1,4,李美宁1,**()
1 山西医科大学生物化学与分子生物学教研室 太原 030001
2 第四军医大学口腔颌面外科国家重点实验室 西安 710032
3 忻州职业技术学院医学系 忻州 034000
4 首都儿科研究所生物技术室 北京 100020
The Preparation and Application of the Ucp2 Gene RNA Probe Labeled by Digoxin
Zhi-zhen LIU1,San-san JIA1,2,Kai-li ZHANG1,Yu-qing SUN1,Hong ZHAO1,Yu-hui HAO3,Bo NIU1,4,Mei-ning LI1,**()
1 Department of Biochemistry & Molecular Biology, Shanxi Medical University, Taiyuan 030001, China;
2 State Key Laboratory of Oral & Maxillofacial Surgery, The Fourth Military Medical University, Xi’an 710032, China;
3 Department of Preclinical Medicine, Xinzhou Vocational and Technical College, Xinzhou 034000, China
4 Laboratory of Biotechnology, Capital Institute of Pediatrics, Beijing 100020, China
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摘要：

目的: 制备用于检测小鼠胚胎早期Ucp2基因表达的地高辛标记的特异性RNA探针。方法: 提取小鼠胚胎脑组织总RNA,设计引物,通过RT -PCR方法获取Ucp2基因片段,将其克隆到pGEM -T载体。分别利用Sp6、T7和Ucp2特异性引物,PCR扩增获得转录模板,通过Sp6及T7 RNA聚合酶,获得地高辛标记的正义、反义Ucp2 RNA原位杂交探针。检测标记探针的效价后,通过全胚胎原位杂交分析制备探针的特异性和杂交效果。结果:成功获得Ucp2基因正义、反义探针,反义探针能高效灵敏检测到Ucp2基因在小鼠胚胎Ed9.5、Ed10.5神经系统呈现高表达,而正义探针未能检测到表达信号。 结论:成功制备了特异高效的地高辛标记Ucp2 RNA 原位杂交探针,为进一步研究Ucp2基因在小鼠胚胎组织中的表达,尤其在神经组织的定位奠定基础。
关键词： Ucp2RNA探针;  全胚胎原位杂交    
Abstract:

Objective: To prepare the Ucp2 gene RNA probe labeled by digoxin, and was used to detect the early Ucp2 gene spatiotemporal expression in mouse embryos. Methods: The primers were designed and the total RNA was extracted from fetal mice nervous tissue. The Ucp2 gene fragment was obtained by RT-PCR and cloned into the pGEM -T vector. Then transcription template was obtained using Sp6, T7 and Ucp2 primers by PCR. Using Sp6 and T7 RNA polymerase, the digoxingenin-labeled antisense and sense probes were synthesized and confirmed by whole embryo in situ hybridization. Results: The plasmid pGEMT-UCP2 was constructed. The digoxingenin-labeled antisense Ucp2 probe was effectively detected in mice Ed9.5 and Ed10.5, and the expression was not detected by the sense probe. Conclusion: The antisense Ucp2 probe can be used specifically and sensitively for study in situ hybridization and lay the foundation for further research of Ucp2 expression in the tissue of mouse embryo, especially in the nerve tissue.
Key words: Ucp2 RNA probe    Whole embryo in situ hybridization
收稿日期: 2018-05-17 出版日期: 2018-10-12
基金资助: * 国家自然科学基金(81741023);山西省回国留学人员科研基金(2016-051)
通讯作者: 李美宁     E-mail: lmnsmu@163.com
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刘志贞
加三三
张凯丽
孙雨晴
赵虹
郝宇卉
牛勃
李美宁

引用本文:

刘志贞,加三三,张凯丽,孙雨晴,赵虹,郝宇卉,牛勃,李美宁. 地高辛标记Ucp2基因RNA探针的制备和应用 *[J]. 中国生物工程杂志, 2018, 38(9): 55-58.

Zhi-zhen LIU,San-san JIA,Kai-li ZHANG,Yu-qing SUN,Hong ZHAO,Yu-hui HAO,Bo NIU,Mei-ning LI. The Preparation and Application of the Ucp2 Gene RNA Probe Labeled by Digoxin. China Biotechnology, 2018, 38(9): 55-58.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180908    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I9/55

图1  重组质粒PCR鉴定
图2  Ucp2 RNA探针电泳图
图3  DIG-Ucp2 RNA探针全胚胎原位杂交
[1] Zaganjor I, Sekkarie A, Tsang B L , et al. Describing the prevalence of neural tube defects worldwide: a systematic literature review. PLoS One, 2016,11(4):e0151586.
doi: 10.1371/journal.pone.0151586
[2] Tissir F, Goffinet A M . Shaping the nervous system: role of the core planar cell polarity genes. Nature Reviews Neuroscience, 2013,14(8):525-535.
doi: 10.1038/nrn3525
[3] Pani L, Horal M, Loeken M R . Rescue of neural tube defects in Pax-3-deficient embryos by p53 loss of function: implications for Pax-3-dependent development and tumorigenesis. Genes & Development, 2002,16(6):676-680.
[4] Shang Y, Zhao H, Niu B , et al. Correlation of polymorphism of MTHFRs and RFC-1 genes with neural tube defects in China. Birth Defects Research Part A: Clinical and Molecular Teratology, 2008,82(1):3-7.
doi: 10.1002/(ISSN)1542-0760
[5] Huang H Y, Chen H L, Feng L P . Maternal obesity and the risk of neural tube defects in offspring: A meta-analysis. Obesity Research & Clinical Practice, 2017,11(2):188-197.
[6] Yang W, Carmichael S L, Tinker S C , et al. Association between weight gain during pregnancy and neural tube defects and gastroschisis in offspring. Birth Defects Research Part A: Clinical and Molecular Teratology, 2012,94(12):1019-1025.
doi: 10.1002/bdra.23057
[7] Lupo P J, Canfield M A, Chapa C , et al. Diabetes and obesity-related genes and the risk of neural tube defects in the national birth defects prevention study. Am[J] Epidemiol. 2012,176(12):1101-1109.
[8] Liu Z, Xie J, Luo T , et al. An epidemiologic study of mitochondrial membrane transportor protein gene polymorphism and risk factors for neural tube defects in Shanxi, China. Neural Regeneration Research, 2012,7(6):463-469.
[9] 孙小慧, 刘晓凡, 赵雅静 , 等. 地高辛标记DNA探针杂交法检测Vero细胞DNA残留量的适用性验证及应用. 微生物学免疫学进展, 2016,44(2):40-44.
Sun X H, Liu X F, Zhao Y J , et al. Applicability validation and application of digoxigenin-labeled DNA probe in detection of residual DNA by hybridization in Vero cell. Progress in Microbiology and Immunology, 2016,44(2):40-44.
[10] Pryor S E, Massa V, Savery D , et al. Convergent extension analysis in mouse whole embryo culture. Methods Mol Biol, 2012,83(9):133-146.
[11] 温宗华, 张艳, 吴自勍 , 等. 地高辛标记斑马鱼cd99l2基因RNA探针的制备. 南方医科大学学报, 2010,30(5):969-972.
Wen Z H, Zhang Y, Wu Z Q , et al. Preparation of RNA probe for cd99l2 gene of zebrafish labeled with digoxingenin-UTP. Journal of Southern Medical University, 2010,30(5):969-972.
[12] 陶春爱, 邱文英, 李刚 , 等. 纳米金标记核酸探针检测小反刍兽疫病毒核酸的研究. 中国生物工程杂志, 2012,32(7):89-94.
Tao C A, Qiu W Y, Li G , et al. Study on peste des petits ruminants virus nucleic acid detection based on gold-nanoparticle conjuncted probe. China Biotechnology, 2012,32(7):89-94.
[13] Lapp D W, Zhang S S, Barnstable C J . Stat3 mediates LIF-induced protection of astrocytes against toxic ROS by upregulating the UPC2 mRNA pool. Glia, 2014,62(2):159-170.
doi: 10.1002/glia.v62.2
[14] Normoyle K P, Kim M, Farahvar A , et al. The emerging neuroprotective role of mitochondrial uncoupling protein-2 in traumatic brain injury. Transl Neurosci, 2015,6(1):179-186.
[15] 蒋骄云, 田文斐, 冯龙 , 等. 地高辛标记的黄鳝F64基因cRNA探针的制备及其应用. 生物技术通报, 2016,32(3):137-141.
Jiang J Y, Tian W F, Feng L , et al. Preparation and application of cRNA probe labeled with digoxingenin for gene F64 of rice field eel. Biotechnology Bulletin, 2016,32(3):137-141.
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