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利用荧光标签的CRISPR敲入技术检测秀丽隐杆线虫中EGL-19的亚型特异性表达。 (注:原文中“in”后面缺少具体内容,这里补充了“秀丽隐杆线虫”使句子完整通顺,你可根据实际情况调整)

Using CRISPR knock-in of fluorescent tags to examine isoform-specific expression of EGL-19 in .

作者信息

McDonald Kara, Larkin Kerry, Dickinson Daniel J, Golden Andy, Bai Xiaofei, Doonan Ryan

机构信息

Glow Worms Stream, Freshman Research Initiative, College of Natural Sciences, The University of Texas at Austin, Austin, Texas, United States.

Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States.

出版信息

MicroPubl Biol. 2023 Sep 7;2023. doi: 10.17912/micropub.biology.000858. eCollection 2023.

Abstract

L-type voltage-gated calcium channels (VGCCs) regulate calcium influx and excitation-contraction coupling in many types of muscle cells. Thus, VGCC mutations can cause skeletal and cardiac muscle diseases in humans, such as Duchenne muscular dystrophy and Timothy syndrome. To better understand the genetics and native expression of VGCCs, we have chosen to use the microscopic roundworm, . The locus is the sole L-type VGCC gene and it encodes three distinct isoforms (a, b, and c). Isoform c is curious because the protein is truncated, lacking the transmembrane domains that form the physical calcium channel. In this study, we have characterized expression using CRISPR/Cas9 genome editing to 'knock-in' fluorescent tags of differing colors, allowing us to distinguish the expression pattern of each isoform. Not surprisingly, we found that EGL-19 is expressed in all types of muscle. In addition, we provide evidence that the truncated c isoform is expressed. Finally, although we find evidence that specific isoforms can have unique subcellular distributions, we also observed some expression patterns that appear to be artifacts. Overall, our results show interesting patterns of expression, but also highlight the need for caution when interpreting expression of reporter genes even when they represent endogenous tags.

摘要

L型电压门控钙通道(VGCCs)调节多种类型肌肉细胞中的钙内流和兴奋-收缩偶联。因此,VGCC突变可导致人类骨骼肌和心肌疾病,如杜氏肌营养不良症和蒂莫西综合征。为了更好地理解VGCCs的遗传学和天然表达,我们选择使用微小的蛔虫。该基因座是唯一的L型VGCC基因,它编码三种不同的亚型(a、b和c)。亚型c很奇特,因为该蛋白质被截断,缺少形成物理钙通道的跨膜结构域。在这项研究中,我们使用CRISPR/Cas9基因组编辑对不同颜色的荧光标签进行“敲入”,从而对该基因的表达进行了表征,使我们能够区分每种亚型的表达模式。不出所料,我们发现EGL-19在所有类型的肌肉中均有表达。此外,我们提供了截断的c亚型表达的证据。最后,尽管我们发现有证据表明特定亚型可具有独特的亚细胞分布,但我们也观察到一些似乎是假象的表达模式。总体而言,我们的结果显示了该基因表达的有趣模式,但也强调了在解释报告基因的表达时即使它们代表内源性标签也需谨慎的必要性。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/aaf7/10514701/247c269fe012/25789430-2023-micropub.biology.000858.jpg

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