使用自切割的 ribozyme 的 Ribo-On 和 Ribo-Off 工具允许操纵 C. elegans 中的内源基因表达
Jie Fang1,2,3, Jie Wang1,2, Yuzhi Wang1,2
1The Fourth Affiliated Hospital, Zhejiang University School of Medicine, 322000, Yiwu, China.
Communications biology
|August 4, 2023
概括
研究人员开发了新的 ribozyme 工具,以精确控制 C. elegans. 中的基因表达. 这些工具使得有效的基因沉默和条件激活成为可能,有助于研究基因功能.
科学领域:
- 分子生物学分子生物学
- 遗传学 是一个遗传学.
- 生物技术是生物技术.
背景情况:
- 研究基因功能需要对内源基因表达进行精确的控制.
- 在"开启"和"关闭"状态之间操纵基因表达的现有方法是有限的.
- 克里斯普尔/卡斯9技术提供了强大的基因组编辑能力.
研究的目的:
- 开发用于有效操纵C. elegans.内源基因表达的新工具.
- 为实现基因沉默和条件基因激活创造方法.
- 扩大用于研究基因功能的工具包.
主要方法:
- 使用CRISPR/Cas9.9将T3H38 ribozyme编码序列插入内源基因的3'未翻译区域 (UTR).
- 开发了一种有条件淘汰系统,通过将loxP侧边的转录终结信号和 ribozyme 插入 3' UTR.
- 利用Cre重组酶进行基因表达的空间或时间控制.
- 采用morpholino注射来阻断 ribozyme 自分裂活性,用于条件基因激活.
主要成果:
- 插入T3H38 ribozyme有效地将内源基因表达减少到几乎无法检测的水平,模仿功能丧失的表型.
- 成功建立了条件淘汰系统,允许通过Cre重组酶对基因表达进行空间或时间控制.
- 条件基因激活是通过用morpholino阻断 ribozyme 活性或通过使用Cre 复合酶去除 ribozyme 磁带来实现的.
结论:
- 基于Ribozyme的工具提供了一种有效的手段,可以在空间和时间上操纵内源基因表达.
- 这些新型工具显著提高了研究基因功能的能力,因为它们能够精确控制基因活动.
- 开发的方法扩大了研究人员调查C. elegans.基因功能的遗传工具包.
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