PUBLICATION
RNaseH-mediated simultaneous piggyback knockdown of multiple genes in adult zebrafish
- Authors
- Raman, R., Ryon, M., Jagadeeswaran, P.
- ID
- ZDB-PUB-201121-4
- Date
- 2020
- Source
- Scientific Reports 10: 20187 (Journal)
- Registered Authors
- Jagadeeswaran, Pudur
- Keywords
- none
- MeSH Terms
-
- Animals
- Embryo, Nonmammalian
- Gene Knockdown Techniques*
- Morpholinos/genetics
- Oligonucleotides, Antisense
- Zebrafish/genetics*
- Zebrafish Proteins/genetics*
- PubMed
- 33214638 Full text @ Sci. Rep.
Citation
Raman, R., Ryon, M., Jagadeeswaran, P. (2020) RNaseH-mediated simultaneous piggyback knockdown of multiple genes in adult zebrafish. Scientific Reports. 10:20187.
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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping