Transposon-mediated targeted and specific knockdown of maternally expressed transcripts in the ascidian Ciona intestinalis

Abstract

Maternal mRNAs play crucial roles during early embryogenesis of ascidians, but their functions are largely unknown. In this study, we developed a new method to specifically knockdown maternal mRNAs in Ciona intestinalis using transposon-mediated transgenesis. We found that GFP expression is epigenetically silenced in Ciona intestinalis oocytes and eggs, and this epigenetic silencing of GFP was used to develop the knockdown method. When the 5′ upstream promoter and 5′ untranslated region (UTR) of a maternal gene are used to drive GFP in eggs, the maternal gene is specifically knocked down together with GFP. The 5′ UTR of the maternal gene is the major element that determines the target gene silencing. Zygotic transcription of the target gene is unaffected, suggesting that the observed phenotypes specifically reflect the maternal function of the gene. This new method can provide breakthroughs in studying the functions of maternal mRNAs.

piRNA-like small RNAs are responsible for the maternal-specific knockdown in the ascidian Ciona intestinalis Type A

“…In a previous study, we showed that both eGFP and Kaede can induce MASK 22 . These reporter genes were driven by the cis elements of targeted maternal genes for inducing MASK.…”

“…In MASK, the location of the MASK vector in the genome is usually not close to the genomic locus where the target gene is encoded 22 . A factor created from the MASK vector is thus suspected to reach the target gene or its mRNA to suppress it.…”

“…Our group recently established a new reverse genetic method for knocking down the maternal expression of genes in the ascidian Ciona intestinalis Type A 22 . The method, named MASK (maternal mRNA-specific knockdown), uses an epigenetic suppression of the maternal expression of reporter genes (such as enhanced green fluorescent protein [ eGFP ] gene) from the cis elements of maternally expressed genes.…”

“…In the transgenic lines, the eGFP expression in oocytes and eggs is somehow silenced by an epigenetic mechanism. When the cis element plus the 5′ untranslated region (5′ UTR) of a maternal gene is used to drive eGFP , the maternal expression of the gene is silenced together with eGFP in oocytes and eggs 22 . Curiously, the zygotic expression of neither eGFP nor the targeted maternal gene (if it has zygotic expression in Ciona ) is silenced by MASK.…”

“…In our previous report about the use of MASK 22 , we induced MASK by eGFP 26 and Kaede 27 reporter genes, suggesting the possibility that multiple reporter genes could be used for MASK. A deeper investigation of the reporter genes that are compatible with MASK is necessary.…”