Delivery of exogenous proteins into eggs by injection into the mother’s ovary (IMO) in zebrafish
Delivery of exogenous proteins into eggs by injection into the mother’s ovary (IMO) in zebrafish
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通过注射到斑马鱼母亲的卵巢（IMO）将外源蛋白质输送到卵中
DOI：
10.1007/s10695-021-00945-6
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发表时间：
2021
影响因子：
2.9
通讯作者：
岩泉雅樹 横井勇人 鈴木徹
中科院分区：
文献类型：
--
作者：
伊藤 玄;旗 薫;北村 淳一;古屋 康則;岩泉雅樹 横井勇人 鈴木徹
基因组编辑对生物实验产生了深远的影响，现在可以应用于许多生物体，包括非传统模型。然而，基因组编辑组件的引入是耗时耗力的，有时还需要特殊的显微注射技术。在这项研究中，我们开发了一种技术，通过注射到母亲的卵巢（IMO）中将外源蛋白质递送到卵子中，这导致CRISPR/Cas9递送到卵生动物（包括鱼类）的卵子中。为了测试这种技术，我们检查了用GFP或荧光素酶（Luc）标记的外源蛋白和荧光标记的RNP（Cas9和sgRNA复合物）是否可以通过IMO递送到鸡蛋中。当通过IMO递送GFP-Luc或Cas9-Luc时，通过GFP荧光或荧光素酶活性证实它们掺入受精卵中;蛋白质在蛋黄中积累。Cas9-RNP（靶向酪氨酸酶）也被掺入到鸡蛋中。然而，尚未观察到靶基因酪氨酸酶的基因组编辑。这可能是因为IMO递送的RNP被包装在卵黄颗粒中，而没有到达胚胎核。因此，该报告显示，包括Cas9-RNP在内的外源分子通过IMO成功地递送到受精卵中。将递送的RNP转移到细胞核中对于通过IMO递送系统成功进行基因组编辑至关重要。
Genome editing has had profound effects on biological experimentation and can now be applied to many organisms, including non-conventional models. However, the introduction of genome editing components is time- and labor-consuming and sometimes requires special skills for microinjection. In this study, we developed a technique to deliver exogenous proteins into eggs by injection into the mother’s ovary (IMO), which leads to the delivery of CRISPR/Cas9 into the eggs of oviparous animals, including fish. To test this technique, we examined whether exogenous proteins tagged with GFP or luciferase (Luc), and fluorescent-labeled RNP (Cas9 and sgRNA complex), can be delivered into eggs by IMO. When GFP-Luc or Cas9-Luc was delivered by IMO, their incorporation into fertilized eggs was confirmed by GFP fluorescence or luciferase activity; proteins were accumulated in the yolk. Cas9-RNP (targetingtyrosinase) was also incorporated into the eggs. However, genome editing of the target gene,tyrosinase, was not observed yet. This is presumably because the RNP delivered by IMO was packed in the yolk granules and did not reach into the embryonic nuclei. Thus, this report shows that exogenous molecules including Cas9-RNP were successfully delivered into fertilized eggs by IMO. Transferring the delivered RNP into nuclei will be critical for successful genome editing via the IMO delivery system.
虹鳟（Oncorhynchus mykiss）卵黄发生过程中卵母细胞生长的动态。
影响因子：
3.6
作者：
C. Tyler;J. Sumpter;P. Witthames
通讯作者：
P. Witthames