Wittig Johannes G, Billmeier Martina, Lozano-Velasco Estefanía, García Miguel Robles-, Münsterberg Andrea E
School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK.
School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK.
Dev Biol. 2019 Jan 15;445(2):163-169. doi: 10.1016/j.ydbio.2018.11.008. Epub 2018 Nov 26.
Studying microRNA networks during heart development is essential to obtain a better understanding of developmental defects and diseases associated with the heart and to identify novel opportunities for therapeutics. Here we highlight the advantages of chicken embryos as a vertebrate model for studying intermediate processes of heart development. Avians develop a four-chambered heart closely resembling human anatomy and they develop ex utero, which makes them easily accessible. Furthermore, embryos are available all year with a steady supply.
In this report we established a novel method for the knockdown of microRNA function by microinjecting AntagomiRs into the chicken heart in ovo. Our approach enables the targeted delivery of antagomirs into a locally restricted area and is not impacted by circulation. After further embryo development the successful miRNA knockdown was confirmed. Loss of function phenotypes can be evaluated rapidly, compared to more time-consuming genetic ablation experiments. The local application avoids potential systemic effects of microRNA knockdown, therefore allowing the assessment of impacts on heart development only. The method can be adjusted for different stages of chicken embryos (HH13-HH18) as well as for knockdown or targeted overexpression of coding genes.
In conclusion our method allows targeted and locally restricted delivery of Antagomirs to the heart leading to successful knockdown of microRNA function. This method enables rapid phenotypic assessment, for example by gene expression analysis of multiple cardiac genes.
研究心脏发育过程中的微小RNA网络对于更好地理解与心脏相关的发育缺陷和疾病以及确定新的治疗机会至关重要。在此，我们强调鸡胚作为研究心脏发育中间过程的脊椎动物模型的优势。鸟类发育出一个与人类解剖结构非常相似的四腔心脏，并且它们在体外发育，这使得它们易于获取。此外，全年都有胚胎供应且供应稳定。
在本报告中，我们建立了一种通过向鸡胚心脏内显微注射抗微小RNA（AntagomiRs）来敲低微小RNA功能的新方法。我们的方法能够将抗微小RNA靶向递送至局部受限区域，且不受循环的影响。在胚胎进一步发育后，证实了微小RNA的成功敲低。与耗时更长的基因消融实验相比，功能缺失表型能够快速得到评估。局部应用避免了微小RNA敲低的潜在全身效应，因此仅允许评估对心脏发育的影响。该方法可针对鸡胚的不同阶段（HH13 - HH18）进行调整，也可用于敲低或靶向过表达编码基因。
总之，我们的方法允许将抗微小RNA靶向并局部受限地递送至心脏，从而成功敲低微小RNA功能。该方法能够进行快速的表型评估，例如通过对多个心脏基因的表达分析。
