利用 CRISPR/Cas9 生成 ints14 基因敲除斑马鱼,用于发育和疾病机制研究。

Development & reproduction Pub Date : 2023-12-01 Epub Date: 2023-12-31 DOI:10.12717/DR.2023.27.4.205
Ji Hye Jung, Sanghoon Jeon, Heabin Kim, Seung-Hyun Jung
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引用次数: 0

摘要

INTS14/VWA9是整合子复合体亚基的一个组成部分,它在调节由RNA聚合酶II转录的大量新生RNA的命运,特别是在小核RNA和增强子RNA的生物发生过程中起着关键作用。尽管其重要性不言而喻,但在发育研究方面一直缺乏一个全面的突变模型。为了填补这一空白,我们旨在研究 INTS14 在斑马鱼胚胎发育过程中的表达模式。我们利用 CRISPR/Cas9 系统生成了 ints14 突变株。我们将 Cas9 蛋白和单条引导 RNA 共同注入受精斑马鱼卵中,验证了 gRNA 的活性,随后确认了 ints14 基因中存在 6 或 9-bp 的缺失。此外,我们还通过 PCR 分析、T7E1 检测、TA 克隆和测序对两个突变等位基因进行了检测。我们首次利用CRISPR/Cas9系统创建了一个删除ints14基因部分序列的模型。这一突破为深入探索ints14在动物疾病中的作用开辟了新途径。本研究中产生的突变株可以为进一步研究斑马鱼发育过程中ints14基因缺失的具体后果提供宝贵的资源。这项研究为今后探索 ints14 功能的分子机制、它与其他基因或蛋白的相互作用及其对生物过程的广泛影响奠定了基础。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Generation of ints14 Knockout Zebrafish using CRISPR/Cas9 for the Study of Development and Disease Mechanisms.

INTS14/VWA9, a component of the integrator complex subunits, plays a pivotal role in regulating the fate of numerous nascent RNAs transcribed by RNA polymerase II, particularly in the biogenesis of small nuclear RNAs and enhancer RNAs. Despite its significance, a comprehensive mutation model for developmental research has been lacking. To address this gap, we aimed to investigate the expression patterns of INTS14 during zebrafish embryonic development. We generated ints14 mutant strains using the CRISPR/Cas9 system. We validated the gRNA activity by co-injecting Cas9 protein and a single guide RNA into fertilized zebrafish eggs, subsequently confirming the presence of a 6- or 9-bp deletion in the ints14 gene. In addition, we examined the two mutant alleles through PCR analysis, T7E1 assay, TA-cloning, and sequencing. For the first time, we used the CRISPR/Cas9 system to create a model in which some sequences of the ints14 gene were removed. This breakthrough opens new avenues for in-depth exploration of the role of ints14 in animal diseases. The mutant strains generated in this study can provide a valuable resource for further investigations into the specific consequences of ints14 gene deletion during zebrafish development. This research establishes a foundation for future studies exploring the molecular mechanisms underlying the functions of ints14, its interactions with other genes or proteins, and its broader implications for biological processes.

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