Biomedical Research Center, Qatar University, Doha, Qatar; Department of Biological and Environmental Sciences, College of Arts and Science, Qatar University, Doha, Qatar.
Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin.
J Vis Exp. 2022 Aug 9(186). doi: 10.3791/63324.
The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression. Morpholinos (MOs) have the advantage in biological stability over DNA oligos because they are not susceptible to enzymatic degradation. For optimal effectiveness, MOs are injected into 1-4 cell stage embryos. The temporal efficacy of knockdown is variable, but MOs are believed to lose their effects due to dilution eventually. Morpholino dilution and injection amount should be closely controlled to minimize the occurrence of off-target effects while maintaining on-target efficacy. Additional complementary tools, such as CRISPR/Cas9 should be performed against the target gene of interest to generate mutant lines and to confirm the morphant phenotype with these lines. This article will demonstrate how to design, prepare, and microinject a translation-blocking morpholino against hand2 into the yolk of 1-4 cell stage zebrafish embryos to knockdown hand2 function and rescue these "morphants" by co-injection of mRNA encoding the corresponding cDNA. Subsequently, the efficacy of the morpholino microinjections is assessed by first verifying the presence of morpholino in the yolk (co-injected with phenol red) and then by phenotypic analysis. Moreover, cardiac functional analysis to test for knockdown efficacy will be discussed. Finally, assessing the effect of morpholino-induced blockage of gene translation via western blotting will be explained.
基于 morpholino 寡聚体的敲低系统已被用于通过缺失或降低表达来鉴定各种基因产物的功能。与 DNA 寡聚体相比，morpholino（MO）在生物稳定性方面具有优势，因为它们不易被酶降解。为了达到最佳效果，将 MO 注射到 1-4 细胞期胚胎中。敲低的时间效果是可变的，但由于稀释，MO 最终会失去效果。应密切控制 MO 的稀释和注射量，以最大程度地减少脱靶效应的发生，同时保持靶效。此外，还应针对感兴趣的靶基因使用 CRISPR/Cas9 等其他互补工具来产生突变系，并使用这些系来确认突变体表型。本文将演示如何设计、准备和微注射针对 hand2 的翻译阻断 morpholino 到 1-4 细胞期斑马鱼胚胎的卵黄中，以敲低 hand2 功能，并通过共注射编码相应 cDNA 的 mRNA 来拯救这些“突变体”。随后，通过验证卵黄中存在 morpholino（与苯酚红共注射），然后通过表型分析来评估 morpholino 微注射的效果。此外，还将讨论用于测试敲低效果的心脏功能分析。最后，将解释通过 Western blot 评估基因翻译阻断的 morpholino 诱导的阻断效果。
