The knockdown efficiency of telomere associated genes with specific methodology in a zebrafish cell line

Zebrafish is broadly used as a model organism in gene loss-of-function studies in vivo, but its employment in vitro is greatly limited by the lack of efficient gene knockdown approaches in zebrafish cell lines such as ZF4. In this article, we attempted to induce silencing of telomere associated genes in ZF4 by applying the frequently-used siRNA transfection technology and a novel moiety-linked morpholino (vivo-MO). By proceeding with integrated optimization of siRNAs transfection and vivo-MOs treatment, we compared five transfection reagents and vivo-MOs simultaneously to evaluate the efficiency of terfa silencing in ZF4. 48 h after siRNAs transfection, LipofectamineTM 3000 and X-tremeGENETM HP leaded to knockdown in 35% and 43% of terfa transcription, respectively, while vivo-MO-terfa modulated 58% down-expression of zfTRF2 in contrast to vivo-MO -ctrl 72 h after treatment. Further siRNAs transfection targeting telomere associated genes by X-tremeGENETM HP showed silencing in 40-68% of these genes without significant cytotoxicity and off-target effect. Our results confirmed the feasibility of gene loss-of function studies in a zebrafish cell line, offered a systematic optimizing strategy to employ gene silencing experiments, and presented LipofectamineTM 3000, X-tremeGENETM HP and vivo-morpholinos as candidate gene silencing approaches for zebrafish in vitro gene loss-of-function studies. Successfully knockdown of shelterin genes further opened a new field for telomeric study in zebrafish. (c) 2021 Published by Elsevier B.V.

Automated summary

In this study, gene silencing was successfully achieved in a zebrafish cell line ZF4. By comparing the effects of siRNA transfection and vivo-MO treatment, potential gene silencing approaches for zebrafish cell lines were identified, providing a systematic optimization strategy for further gene loss-of-function studies.