| 何蓉蓉 丁雪燕 陈丹峰 宋婧文 荆清.分子信标用于活细胞内源性mRNA 检测的实验研究[J].,2016,16(17):3227-3231 |
| 分子信标用于活细胞内源性mRNA 检测的实验研究 |
| Experimental Research on Molecular Beacons used of Detection ofEndogenous mRNA in Living Cells |
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| DOI： |
| 中文关键词: 分子信标(MB) 内源性 熔解曲线 SLO 斑马鱼 |
| 英文关键词: Molecular beacons (MB) Endogenous Melting curves SLO Zebrafish |
| 基金项目:国家自然科学基金面上项目（81470407） |
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| 中文摘要: |
| 目的：观察分子信标（molecular beacons，MB）在活细胞内及在体水平对内源性mRNA 检测的可行性。方法：合成增强型绿色
荧光蛋白(Enhanced Green Fluorescent Protein，eGFP)的MB，模拟体内条件，分别从MB 的稳定性、与目标基因结合的特异性和敏
感性进行检测，并通过在活细胞和斑马鱼胚胎内的应用来观察其对内外源性mRNA的检测的可行性。结果：MB 在37℃条件下在
体外48 小时内经琼脂糖凝胶电泳鉴定未见降解，熔解曲线结果显示荧光强度差异无统计学意义（P>0.05）；并且只有在靶序列存
在的条件下MB 的信号强度显著增强，MB 的信号强度与靶基因浓度呈正相关并能基本稳定存在；经eGFP 和靶向eGFP 的MB
共转染的293T细胞能看到荧光表达，靶向eGFP 的MB可特异性结合细胞内eGFP 的mRNA；在单细胞期斑马鱼胚胎内共注射
MB 以及目标单链后能观察到荧光表达。结论：结果表明分子信标可用于活细胞内内源性mRNA的检测。 |
| 英文摘要: |
| Objective:To expand the application of molecular beacons (MB) to detect endogenous mRNAin living cells.Methods:As we have the transgenic zebrafish line containing eGFP, we synthetized the eGFP MB. We first tested the stability and sensitivity of
eGFP MB under the in vitro conditions simulated like in vivo. We then checked the specificity of this MB through binding with synthetized
target sequence assay. We further explore its use in living cells and zebrafish embryos respectively by transfection and microinjection.Results:The eGFP MB was stable under the experimental conditions（P>0.05）and could specifically bind to target gene. The fluorescence
of MB could not be detected with low concentration of target gene. Living 293T cells have fluorescence expression after transfection
of eGFP and eGFP MB. The fluorescence of MB also could be observed in 1-cell stage zebrafish embryos injected with eGFP
MB.Conclusion:The results showed that the molecular beacons can be used in the detection of genes within living cells, and its application
in zebrafish embryos is promising. |
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