N-terminally endogenous mKate2-tagged HIM-4 in Caenorhabditis elegans.
N-terminally endogenous mKate2-tagged HIM-4 in Caenorhabditis elegans.
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DOI：
10.17912/micropub.biology.000386
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发表时间：
2021-04-29
影响因子：
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通讯作者：
Ihara S
中科院分区：
文献类型：
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作者：
Ihara S
基因him-4编码秀丽隐杆线虫的半球蛋白同源物，半球蛋白是一种进化上保守的细胞外基质蛋白。尽管是一种细胞外基质蛋白，但在发育过程中，他-4的突变会导致多性缺陷，并表现出组织脆弱性。虽然之前在秀丽隐杆线虫中的研究已经证实了转基因HIM-4::GFP或内源性c末端标记的HIM-4::mNeonGreen的定位，但秀丽隐杆线虫群落中只有绿色或绿/黄荧光蛋白标记的HIM-4。在这里，我使用CRISPR/Cas9技术将远红色荧光蛋白mKate2插入到him-4基因组位点，建立表达mKate2:: him-4的蠕虫。正如预期的那样，在L4期观察到mkate2:: hm -4在脊柱的定位。与基底膜主要成分IV型胶原或氮原的定位不同，mkate2:: hm -4在咽基底膜前部极化。咽基底膜在L1-L4期保持着独特的极化定位模式。
The gene him-4 encodes the C. elegans homolog of hemicentin, an evolutionarily conserved extracellular matrix protein. Despite being an extracellular matrix protein, mutations in him-4 result in pleiotropic defects during development and demonstrate tissue fragility. While previous studies in C. elegans have confirmed the localization of either transgenic HIM-4::GFP or endogenous C-terminally tagged HIM-4::mNeonGreen, only green or green/yellow fluorescent protein-tagged HIM-4 are available to the C. elegans community. Here, I used CRISPR/Cas9 technology to insert the far-red fluorescence protein mKate2 at the him-4 genomic locus, and established worms expressing mKate2::HIM-4. As expected, localization of mkate2::HIM-4 at the rachis was observed at the L4 stage. In contrast to the localization of type IV collagen or nidogen such as major components of the basement membrane, mkate2::HIM-4 was polarized at the anterior part of the pharyngeal basement membrane. A unique polarized localization pattern of pharyngeal basement membrane is maintained throughout the L1–L4 stages.