地高辛标记Nkx2.1基因RNA探针的制备及应用
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摘要
目的制备地高辛(digoxigenin,DIG)标记的Nkx2. 1基因RNA探针,用于小鼠胚胎早期神经管发育关键时期Nkx2. 1基因表达的检测。方法巢式PCR法扩增Nkx2. 1基因,将其克隆至pGEM-T载体;以其为模板,通过Nkx2. 1特异性引物,PCR扩增RNA探针转录模板;采用T7 RNA聚合酶,制备DIG标记的正义、反义Nkx2. 1 RNA原位杂交探针。经全胚胎原位杂交(whole-mount in situ hybridization,WM-ISH)技术分析胚胎8. 5、9. 5、10. 5 d小鼠体内Nkx2. 1基因的表达情况。结果制备的探针浓度为1 521. 986 ng/mL,纯度A260/280=2. 39。Nkx2. 1基因反义探针能高效检测到Nkx2. 1基因在胚胎8. 5、9. 5及10. 5 d小鼠神经系统中表达,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DIG标记的Nkx2. 1基因RNA原位杂交探针,为进一步研究Nkx2. 1基因在小鼠胚胎组织中的表达,探索其在神经管发育中的作用机制奠定了基础。
Objective To prepare digoxin(DIG)-labeled Nkx2. 1 gene RNA probe and apply to the determination of expression of Nkx2. 1 gene at the key stage of early neural tube development in mouse embryos. Methods Nkx2. 1 gene was amplified by nested PCR and cloned into vector pGEM-T. The constructed recombinant plasmid was used as a template for amplification of transcription template of RNA probe by PCR using Nkx2. 1 specific primers. The DIG-labeled sense and antisense Nkx2. 1 RNA in situ hybridization probes were prepared with T7 RNA polymerase. The expressions of Nkx2. 1 gene in mouse embryos at ages of 8. 5,9. 5 and 10. 5 d were analyzed whole-mount in situ hybridization(WMISH). Results The concentration and purity(A260/280) of the prepared probe were 1 521. 986 ng/m L and 2. 39 respectively. The expression of Nkx2. 1 gene was effectively detected in the mouse embryos at ages of 8. 5,9. 5 and 10. 5 d by Dig-labeled antisense Nkx2. 1 probe,but not detected by the sense probe. Conclusion Specific and effective DIG-labeled Nkx2. 1 gene RNA in situ hybridization probe was prepared successfully,which laid a foundation of further research of Nkx2. 1 expression in the mouse embryo tissue and the mechanism of the gene in neural tube development.
Objective To prepare digoxin(DIG)-labeled Nkx2. 1 gene RNA probe and apply to the determination of expression of Nkx2. 1 gene at the key stage of early neural tube development in mouse embryos. Methods Nkx2. 1 gene was amplified by nested PCR and cloned into vector pGEM-T. The constructed recombinant plasmid was used as a template for amplification of transcription template of RNA probe by PCR using Nkx2. 1 specific primers. The DIG-labeled sense and antisense Nkx2. 1 RNA in situ hybridization probes were prepared with T7 RNA polymerase. The expressions of Nkx2. 1 gene in mouse embryos at ages of 8. 5,9. 5 and 10. 5 d were analyzed whole-mount in situ hybridization(WMISH). Results The concentration and purity(A260/280) of the prepared probe were 1 521. 986 ng/m L and 2. 39 respectively. The expression of Nkx2. 1 gene was effectively detected in the mouse embryos at ages of 8. 5,9. 5 and 10. 5 d by Dig-labeled antisense Nkx2. 1 probe,but not detected by the sense probe. Conclusion Specific and effective DIG-labeled Nkx2. 1 gene RNA in situ hybridization probe was prepared successfully,which laid a foundation of further research of Nkx2. 1 expression in the mouse embryo tissue and the mechanism of the gene in neural tube development.
引文
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[11]EBERLE A B.Spike-in normalization of ChIP data using DNA-DIG-antibody complex[J].Methods Mol Biol,2018:227-238.
[2]ZHANG H N,GUO Y,GU H,et al.TRIM4 is associated with neural tube defects based on genome-wide DNA methylation analysis[J].Clin Epigenetics,2019,11(17):1-13.
[3]ZAGANJOR I,SEKKARIE A,TSANG B L,et al.Describing the prevalence of neural tube defects worldwide:a systematic literature review[J].Plos One,2016,11(4):e0151586.
[4]YU J,MU J B,GUO Q,et al.Transcriptomic profile analysis of mouse neural tube Development by RNA-Seq[J].IUBMBLife,2017,69(9):706-719.
[5]SUN X H,LIU X F,ZHAO Y J,et al.Applicability verification and application of digoxin labeled DNA probe hybri-dization for detection of residual DNA residues in Vero cells[J].Prog Microbiol Immunol,2016,44(2):40-44.(in Chinese)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[J].微生物学免疫学进展,2016,44(2):40-44.
[6]WEI Q,MANLEY N R,CONDIE B G.Whole mount in situ hybridization of E8.5 to E11.5 mouse embryos[J].J Vis Exp,2011,29(56):e2797-e2797.
[7]CONLON R A,HERRMANN B G.Detection of messenger RNAby in situ hybridization to postimplantation embryo whole mounts[J].Methods Enzymol,1993,225(1):373-383.
[8]JIANG J Y,TIAN W F,FENG L,et al.Preparation and application of digoxin-labeled astragalus F64 gene cRNA probe[J].Biotechnol Bull,2016,32(3):137-141.(in Chinese)蒋骄云,田文斐,冯龙,等.地高辛标记的黄鳝F64基因c RNA探针的制备及其应用[J].生物技术通报,2016,32(3):137-141.
[9]HAO J T,WANG S,JIANG W.Molecular mechanism of small activating RNA up-regulating epithelial cadherin expression in malignant tumors and its biological significance[J].Chin JCancer Biother,2019,26(1):115-121.(in Chinese)郝家涛,王帅,蒋伟.小激活RNA上调上皮钙黏蛋白在恶性肿瘤中表达的分子机制及其生物学意义[J].中国肿瘤生物治疗杂志,2019,26(1):115-121.
[10]TAO C A,QIU W Y,LI G,et al.Study on detection of nucleic acid of Peste des Petits Ruminants virus by nanogoldlabeled nucleic acid probe[J].Chin J Bioengi,2012,32(7):89-94.(in Chinese)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[J].中国生物工程杂志,2012,32(7):89-94.
[11]EBERLE A B.Spike-in normalization of ChIP data using DNA-DIG-antibody complex[J].Methods Mol Biol,2018:227-238.