RNaseH-mediated Simultaneous Piggyback Knockdown of Multiple Genes in Adult Zebrafish

Overview

Journal Sci Rep

Specialty Science

Date 2020 Nov 20

PMID 33214638

Citations 4

Authors

Revathi Raman

Mia Ryon

Pudur Jagadeeswaran

Affiliations

Soon will be listed here.

Abstract

We recently developed a piggyback knockdown method that was used to knockdown genes in adult zebrafish. In this method, a vivo morpholino (VMO) piggybacks an antisense deoxyoligonucleotide (dO) into the somatic cells and reduces the cognate mRNA levels. In this paper, we tested whether we can piggyback more than one dO with one VMO. We designed various hybrids that had more than one dO that could be piggybacked with one VMO. We chose f7, f8, and αIIb genes and tested their knockdown by the appropriate assays. The knockdown with piggybacking either two or three dOs by one VMO yielded > 85% knockdown efficiency. We also performed knockdown of argonautes and rnaseh separately along with f7. We found the knockdown of f7 occurs when knockdown of argonautes happens and not when rnaseh knockdown was performed, suggesting that RNaseH is involved in mRNA degradation. In conclusion, we developed a method where we could knockdown three genes at one time, and by increasing the concentration of VMO by twofold, we could knockdown six genes simultaneously. These multiple gene knockdowns will not only increase the efficiency of the method in whole genome-wide knockdowns but will also be useful to study multifactorial disorders.

Citing Articles

Piggyback knockdown screening of unique genes of zebrafish young thrombocytes identifies eight novel genes in thrombopoiesis.

Fallatah W, Mary J, Dhinoja S, Vallabhaneni S, Jagadeeswaran P Sci Rep. 2025; 15(1):5180.

PMID: 39939668 PMC: 11822207. DOI: 10.1038/s41598-025-88866-9.

Knockdown of zebrafish tmem242 enhances the production of ROS that signals to increase f9a expression resulting in DIC-like condition.

Deebani A, Mary J, Dhinoja S, Al Qaryoute A, Fallatah W, Jagadeeswaran P Sci Rep. 2025; 15(1):3058.

PMID: 39856164 PMC: 11761473. DOI: 10.1038/s41598-025-87117-1.

Studying the Role of HOX Genes in Thrombocyte Development.

Mary J, Jagadeeswaran P Methods Mol Biol. 2025; 2889:107-119.

PMID: 39745608 DOI: 10.1007/978-1-0716-4322-8_8.

Pancytopenia and thrombosis defects in zebrafish mutants of Fanconi anemia genes.

Raman R, Ramanagoudr-Bhojappa R, Dhinoja S, Ramaswami M, Carrington B, Jagadeeswaran P Blood Cells Mol Dis. 2022; 93:102640.

PMID: 34991062 PMC: 8760166. DOI: 10.1016/j.bcmd.2021.102640.

Knockdown screening of chromatin binding and regulatory proteins in zebrafish identified Suz12b as a regulator of tfpia and an antithrombotic drug target.

Raman R, Fallatah W, Al Qaryoute A, Dhinoja S, Jagadeeswaran P Sci Rep. 2021; 11(1):15238.

PMID: 34315984 PMC: 8316476. DOI: 10.1038/s41598-021-94715-2.

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