Method of inducible knockdown of essential genes in osc cell culture of Drosophila melanogaster

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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.

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About the authors

S. V. Marfina

National Research Center “Kurchatov Institute”; Mendeleev University of Chemical Technology

Email: n.akulenko11@gmail.com
Russian Federation, Moscow, 123182; Moscow, 125047

E. A. Mikhaleva

National Research Center “Kurchatov Institute”

Email: n.akulenko11@gmail.com
Russian Federation, Moscow, 123182

N. V. Akulenko

National Research Center “Kurchatov Institute”

Author for correspondence.
Email: n.akulenko11@gmail.com
Russian Federation, Moscow, 123182

S. S. Ryazansky

National Research Center “Kurchatov Institute”

Email: s.ryazansky@gmail.com
Russian Federation, Moscow, 123182

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Supplementary files

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2. Fig. 1. Results of Cul3 and EloB gene knockout in OSC. Changes in the expression level of Cul3 and EloB genes in knockout cells relative to the original OSC[Cas9+] line (a). The expression level measured by qRT-PCR was normalized to rp49 expression. The average measurement values ​​for two biological replicates with three technical replicates each are shown; the measurement errors are shown as the standard error of the mean. The statistical significance of differences was assessed using the Wilcoxon method. PCR analysis of the genome of Cul3-KO (b) and EloB-KO (c) cells revealed the presence of both wild-type alleles and a mutant allele with an insertion of the hyg gene in both lines. The arrangement of the primers used for the gene sequence, the size of the amplicons, and the PCR result (electrophoresis in an agarose gel) are shown.

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3. Fig. 2. Scheme of construction of donor plasmid pAc-MT-shRNA-bsd containing MT-shRNA-bsd cassette with inducible shRNA (a). Detailed description is presented in the text and Experimental section. puro_R and puro_L are fragments of puro gene in pAc-MT-shRNA-bsd which will serve as arms for homologous recombination. MT-shRNA-bsd cassette from donor plasmid is introduced into pAc-sgRNA-Cas9 transgene preliminarily integrated into genome (its part with puro and Cas9 genes is shown) using Cas9 and is activated by addition of copper ions (b). Expressed shRNAs are processed to siRNAs which in complex with Drosophila Ago2 protein bind to complementary mRNA and cause its cleavage by RNAi mechanism.

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4. Fig. 3. Changes in the expression level of the Cul3 (a) and cut (b) genes upon addition of copper ions at concentrations of 0.5, 1, and 2 mM to cell lines in which cassettes with the corresponding shRNAs were introduced into the genome, compared to expression in OSC[Cas9+]. The expression level measured by qRT-PCR was normalized to the expression of rp49. Shown are the average values ​​of measurements for two to three biological replicates with three technical replicates in each; measurement errors are shown as standard errors of the mean. The statistical significance of differences was assessed using the Wilcoxon method; ns ‒ differences in means are statistically insignificant.

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