Method of inducible knockdown of essential genes in osc cell culture of <i>Drosophila melanogaster</i>

Abstract

In the paper, we propose an RNA interference-based method of inducible knockdown of genes essential for cell viability. The method arranges a genetic cassette in which an inducible metallothionein promoter controls the expression of siRNA precursor. The cassette is inserted into the genomic pre-integrated transgene by CRIPSR-Cas9. The expression of siRNA precursor and following silencing of the gene of interest is activated by the supplementation of the medium with copper ions. This technique with the production of endogenous siRNAs allows the gene knockdown in cell cultures that are refractory to conventional transfection strategies of exogenous siRNA. The efficiency of the developed method was demonstrated in the cell culture of Drosophila ovarian somatic cells for two genes that are essential for oogenesis: Cul3, encoding a component of the multiprotein ubiquitin-ligase complex with versatile functions in proteostasis, and cut, encoding a transcription factor regulating the differentiation of the ovarian somatic cells.