Isolation of Embryonic Cardiomyocytes and Cell Proliferation Assay Using Genetically Engineered Reporter Mouse Model.
Isolation of Embryonic Cardiomyocytes and Cell Proliferation Assay Using Genetically Engineered Reporter Mouse Model.
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DOI：
10.21769/bioprotoc.4802
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发表时间：
2023-09-05
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影响因子：
0.8
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先天性心脏病（CHD）通常与肌源性缺陷有关。在心脏发育过程中，心肌细胞的生长需要来自外部因素的重要线索，如胰岛素样生长因子2（IGF-2）。为了确定生长因子是否以及如何解释胚胎心肌细胞增殖，分离培养胚胎心肌细胞可以用作心脏发育研究的有用工具。目前用于从心脏分离心肌细胞的方案不包括心肌细胞特异性报告物以区分心肌细胞与其他细胞类型。为了优化心肌细胞增殖的可视化，我们的方案利用Tnnt 2启动子驱动的H2 B-GFP敲入小鼠模型（TNNT 2 H2 B-GFP/+）来体外可视化核标记的心肌细胞特异性荧光。心肌细胞特异性遗传报告基因与有效的增殖试验相结合，通过提高细胞鉴定、增殖细胞计数和心肌细胞跟踪的准确性，提高了机制研究的重现性。 该方案改进了先前的心肌细胞分离方法，以特异性靶向胚胎心肌细胞。·使用Yan等人（2016）鉴定的H2 B-GFP/+心肌细胞报告基因。·用磷酸化组蛋白3（p-H3）测定追踪细胞增殖。·在评估生长因子在心肌细胞增殖中的作用方面具有应用。
Congenital heart disease (CHD) is often associated with myogenic defects. During heart development, cardiomyocyte growth requires essential cues from extrinsic factors such as insulin-like growth factor 2 (IGF-2). To determine whether and how growth factors account for embryonic cardiomyocyte proliferation, isolation followed by culturing of embryonic cardiomyocytes can be utilized as a useful tool for heart developmental studies. Current protocols for isolating cardiomyocytes from the heart do not include a cardiomyocyte-specific reporter to distinguish cardiomyocytes from other cell types. To optimize visualization of cardiomyocyte proliferation, our protocol utilizes a Tnnt2-promoter-driven H2B-GFP knock-in mouse model (TNNT2H2B-GFP/+) for in vitro visualization of nuclear-tagged cardiomyocyte-specific fluorescence. A cardiomyocyte-specific genetic reporter paired with an effective proliferation assay improves the reproducibility of mechanistic studies by increasing the accuracy of cell identification, proliferated cell counting, and cardiomyocyte tracking. Key features • This protocol refines previous methods of cardiomyocyte isolation to specifically target embryonic cardiomyocytes. • UsesH2B-GFP/+cardiomyocyte reporters as identified by Yan et al. (2016). • Traces cell proliferation with Phospho-Histone 3 (p-H3) assay. • Has applications in assessing the role of growth factors in cardiomyocyte proliferation.