Molecular and Phenotypic Characterization Following RNAi Mediated Knockdown in Drosophila

Abstract

Loss of function studies shed significant light on the involvement of a gene or gene product in different cellular processes. Short hairpin RNA (shRNA) mediated RNA interference (RNAi) is a classical yet straightforward technique frequently used to knock down a gene for assessing its function. Similar perturbations in gene expression can be achieved by siRNA, microRNA, or CRISPR-Cas9 methods also. In Drosophila genetics, the UAS-GAL4 system is utilized to express RNAi and make ubiquitous and tissue-specific knockdowns possible. The UAS-GAL4 system borrows genetic components of S. cerevisiae, hence rule out the possibility of accidental expression of the system. In particular, this technique uses a target-specific shRNA, and the expression of the same is governed by the upstream activating sequence (UAS). Controlled expression of GAL4, regulated by specific promoters, can drive the interfering RNA expression ubiquitously or in a tissue-specific manner. The knockdown efficiency is measured by RNA isolation and semiquantitative RT-PCR reaction followed by agarose gel electrophoresis. We have employed immunostaining procedure also to assess knockdown efficiency. RNAi provides researchers with an option to decrease the gene product levels (equivalent to hypomorph condition) and study the outcomes. UAS-GAL4 based RNAi method provides spatio-temporal regulation of gene expression and helps deduce the function of a gene required during early developmental stages also.

Keywords: Drosophila melanogaster; Nuclear pores; Nucleoporins; RNAi; Semiquantitative PCR; UAS-Gal4; dElys.

Figure 1.. Schematic diagram detailing the functioning of the UAS-GAL4 system in Drosophila

Figure 2.. A flow chart with details of the protocol

Figure 3.. Steps in the isolation of Salivary glands from Drosophila third instar larva.

Upper panels (a-f), Steps in larva separation and cleaning; (a) Larvae and Pupae in food vial, (b) Squirting water and suspending the top layer of food and larva with a brush, (c) Decanting suspension in a petri-plate, (d) Fly food and third instar larva suspension, (e) Cleaned larva collected in distilled water, (f) A third instar larva. Lower panels (g-l) Steps in larval dissection and salivary gland isolation (g) A third instar larva, (h) Holding a third instar larva with forceps, (i) Pulled out salivary glands and abdominal parts of the third instar larva, (j) Head complex having the brain, imaginal disc, and salivary gland (k) Salivary gland with associated fat bodies, (l) Dissected and cleaned up pair of salivary glands with an attached mouth hook.

Figure 4.. Semiquantitative PCR based assessment of knockdown

. cDNAs prepared from Control and nucleoporin knockdown used in a PCR reaction. Upper panels show levels of Nup160 (A), Nup107 (B), and Sec13 (C) from control and knockdown tissues. RpL49 served as an internal loading control between control and knockdown samples.

Figure 5.. RNAi mediated dElys knockdown depleted dElys signals from salivary gland nuclei.

The salivary glands dissected from Control RNAi (upper panels) and dElys RNAi (lower panels) third instar larvae and stained with dElys (red, first panels), mAb414 (green, second panels), DAPI (blue, third panels). The fourth panels represent the merged images. Scale bars =10 μm.

Figure 6.. Knockdown of Nup160 in eyes and wings induces developmental defects.

(A) Eye specific knockdown of Nup160 using Ey-GAL4 and visualization of the compound eye under bright field light microscope and SEM. (B) Wing specific knockdown of Nup160 using Wg-GAL4 and visualization of wings under bright field light microscope. The control knockdown is for comparison. Scale bars (A) are 200 μm in the left panels, 20 μm in the middle panels, and 2 μm in the right panels. The scale bar in (B) is 200 μm.