Abstract
Antisense oligonucleotides (ASOs) are synthetic, single-strand RNA-DNA hybrids that induce catalytic degradation of complementary cellular RNAs via RNase H. ASOs are widely used as gene knockdown reagents in tissue culture and in Xenopus and mouse model systems. To test their effectiveness in zebrafish, we targeted 20 developmental genes and compared the morphological changes with mutant and morpholino (MO)-induced phenotypes. ASO-mediated transcript knockdown reproduced the published loss-of-function phenotypes for oep, chordin, dnd, ctnnb2, bmp7a, alk8, smad2 and smad5 in a dosage-sensitive manner. ASOs knocked down both maternal and zygotic transcripts, as well as the long noncoding RNA (lncRNA) MALAT1. ASOs were only effective within a narrow concentration range and were toxic at higher concentrations. Despite this drawback, quantitation of knockdown efficiency and the ability to degrade lncRNAs make ASOs a useful knockdown reagent in zebrafish.
Highlights
One effective strategy for interrogating gene function is to disrupt the generation of a gene product by knockdown or knockout
We found that the level of oep mRNA knockdown across individual embryos at shield stage was in line with the variability in phenotypes at 24 hpf (7/21 strong oep phenotype, 11/21 dead, 2/21 partial oep phenotype, 1/21 deformed, versus 13/15 Antisense oligonucleotides (ASOs)-injected embryos with a >3-fold reduction in oep mRNA levels) (Fig 2C)
This study reveals that ASOs can be an effective RNA knockdown reagent for zebrafish
Summary
One effective strategy for interrogating gene function is to disrupt the generation of a gene product by knockdown or knockout. Knockout technologies, such as CRISPR/Cas and homologous recombination, alter the DNA locus of the gene by either introducing a premature stop codon or removing the entire locus (Fig 1A) [1,2]. On the other hand, such as RNAi, siRNAs and modified antisense oligonucleotides [3,4], target the mRNA rather than alter the DNA. While it is most reliable to infer gene function by generating a mutant organism, knockdown reagents can provide a more immediate assessment of gene function and can be used to target gene products without disrupting regulatory DNA elements.
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