Medical College of Wisconsin
CTSICores SearchResearch InformaticsREDCap

Design and Microinjection of Morpholino Antisense Oligonucleotides and mRNA into Zebrafish Embryos to Elucidate Specific Gene Function in Heart Development. J Vis Exp 2022 Aug 09(186)

Date

08/30/2022

Pubmed ID

36036621

Pubmed Central ID

PMC10388372

DOI

10.3791/63324

Scopus ID

2-s2.0-85136787659 (requires institutional sign-in at Scopus site)   1 Citation

Abstract

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.

Author List

Zakaria ZZ, Eisa-Beygi S, Benslimane FM, Ramchandran R, Yalcin HC

Author

Ramani Ramchandran PhD Professor in the Pediatrics department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Animals
Embryo, Nonmammalian
Gene Knockdown Techniques
Morpholinos
Oligonucleotides, Antisense
Phenotype
RNA, Messenger
Zebrafish
Zebrafish Proteins