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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 attempt 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 hours after siRNAs transfection, Lipofectamine™ 3000 and X-tremeGENE™ HP leaded to knockdown in 35% and 43% of terfa mRNA, respectively, while vivo-MO-terfa modulated 58% down-expression of zfTRF2 in contrast to vivo-MO-ctrl 72 hours after treatment. Further siRNA transfection targeting telomere associated genes by X-tremeGENE™ 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 Lipofectamine™ 3000, X-tremeGENE™ 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. Copyright © 2021. Published by Elsevier B.V.
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