Authors: Leonie Misquitta, Qin Wei and Bruce M. Paterson
This protocol was adapted from “Targeted Disruption of Gene Function in Drosophila by RNA Interference,” Chapter 19, in Drosophila Protocols (eds. Sullivan et al.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2000. Please note that this version of the protocol is a 2007 revision.
RNA interference (RNAi) is a powerful method for determining the role of specific genes during Drosophila embryogenesis. This protocol describes a technique by which Drosophila embryos can be injected with dsRNA in order to disrupt targeted gene function. The approach is straightforward, utilizing improved methods for injecting the dsRNA directly through the chorion of the embryo. This strategy minimizes problems normally associated with desiccation of the dechorionated embryo and facilitates post-injection analysis of gene expression.
The double-stranded RNA needed for RNAi is prepared as in Preparation of Double-Stranded RNA for Drosophila RNA Interference (RNAi). Embryos are collected shortly before injection (typically, 30-60 min prior; see Collection of Drosophila Embryos for RNA Interference (RNAi)). This injection technique can also be used for generating transgenic Drosophila lines, as in Drosophila RNA Interference (RNAi) Using a Gal-4 Inducible Transgene Vector (Misquitta et al. 2008). These procedures assume that the investigator has access to a working fly facility and is familiar with basic methods for injection of embryos and the analysis of gene expression in Drosophila embryos. If not, details for these procedures can be found elsewhere; see Ashburner (1989a,b); for microinjection procedures, see Kiehart et al. (2000).
Preparing the Needles
Calibrating the Transjector
3.Set the Transjector to automatic injection.
4.Set the injection pressure (Po) to 450.
5.Set the compensation pressure (Pc) to 570.
6.Set the time of injections to 0.1 sec.
Based on the volume loaded into the needle (0.5-1.0 μL), each embryo receives up to 100-200 pL of dsRNA. A typical loading, if the needle remains unbroken and unblocked, is good for the injection of approximately 1000 embryos or more.
Inject the embryos in the posterior end, slightly off-center, because the posterior tip of the chorion is very hard and needles often break in this position. Keep the injection room at 18°C to slow development.
7.Break the filled needle against the edge of the slide containing the embryos to create a sharp point, as follows:
8.Bring the needle tip and the posterior tip of the embryo into the same focal plane. Make sure that the needle is positioned off-center with respect to the posterior tip of the embryo (see Fig. 1).
Figure 1. Correct injection position for the embryos. (A) Do not inject the embryos directly in the posterior center of the chorion, as the needles often break at this site due to the apparent increased hardness of the chorion. (B) Embryos are injected in the posterior end, slightly off-center, with much less needle breakage or blockage.
For best results, inject the embryos with the needle at an angle slightly greater than 45° relative to the embryo surface in the posterior quarter of the embryo.
9.Insert the needle just far enough to penetrate the vitelline membrane.
10.Inject the RNA into the embryo. A small amount of food dye will appear as a small dot in the posterior end of the embryo. It takes ~1-5 sec to inject each embryo.
Collecting Injected Embryos for Analysis
12.Wash the embryos off the slide into a nylon-mesh collection basket.
13.Transfer the embryos to a microcentrifuge tube.
14.Fix the embryos and stain them using standard protocols; see Ashburner (1989a,b).
The microinjector we use is the Eppendorf Transjector. Although primarily used for the injection of cultured cells, the Transjector is easily adapted for the injection of Drosophila embryos directly through the chorion. Highly reproducible injection conditions can be defined with regard to the volume of material injected, while preventing backflow of material into the needle, which results in needle blockage. This is accomplished by varying the injection pressure, compensation pressure, and the time of injection. The Transjector was a key factor in being able to routinely inject approximately 500 embryos for each analysis. It also has a useful “clean button” that uses a burst of high-pressure air to clean the needle tip in case of blockage. This greatly reduces the number of times needles need to be changed.
In our studies, typical efficiencies for generation of the mutant phenotype in the embryo ranged from 72% to 86%, but penetration of the white mutation to the adult eye was <3% (Misquitta and Paterson 1999). RNAi has also been used in dechorionated embryos to demonstrate that both frizzled and frizzled 2 were in the wingless pathway (Kennerdell and Carthew 1998). However, dechorionated embryos have a much lower survival rate and injection artifacts are more pronounced. To rescue injected transgenic lines, apply a small amount of yeast paste to the slide and transfer the embryos from the paste to food vials. Recent experience has shown that using <1 mg/mL of dsRNA for dsRNAs longer than 1 kb minimizes needle blockage while 1 mg/mL is suitable for dsRNAs shorter than 1 Kb in length.