Protocol

Delivery of dsRNA into Drosophila Embryos by Gene Gun

This protocol was adapted from "RNAi in Drosophila" contributed by Richard W. Carthew, Chapter 17, in RNAi, A Guide to Gene Silencing (ed. Hannon). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

INTRODUCTION

In this method, subcellular-sized particles are accelerated to high velocity to carry double-stranded RNA (dsRNA) into Drosophila embryos. The major advantage of this procedure over microinjection (Microinjection of dsRNA into Drosophila Embryos) is that particle bombardment is easier and faster to perform. In addition, the mechanical trauma received is far less than by microinjection, allowing better survival of embryos and fewer phenotypic artifacts. The principle of particle bombardment is to employ a high-velocity stream of helium gas to accelerate gold particles coated with dsRNA to sufficient velocities to penetrate embryos. This protocol describes the use of the Helios Gene Gun system for particle delivery. Other systems that work with Drosophila embryos include the Biolistic PDS-1000 from Bio-Rad.

MATERIALS

Reagents

Bleach (50%) with H₂O

Calcium chloride (CaCl₂) (1 M)

Drosophila embryos

dsRNA or siRNA (see Synthesis of dsRNA for RNAi in Drosophila: Plasmid Template Method, Synthesis of dsRNA for RNAi in Drosophila: PCR Template Method, and Synthesis of siRNA for RNAi in Drosophila)

Ethanol (absolute)

Gold particles (1.0-µm diameter) (Bio-Rad)

Halocarbon oil

Purchase halocarbon oil Series 700, CAS# 9002-83-9 from Halocarbon Products Corporation, New Jersey.

Polyvinylpyrrolidone (360 kD) (Sigma)

Rubber glue

Spermidine (0.05 M)

Tape glue

Equipment

Aluminum rod frame

Helios Gene Gun (Bio-Rad)

Helium tank

Use compressed helium of grade 4.5 or higher and pressurized to 2600 psi.

Microfuge tube

Microslide

Paint brush (fine-bristle)

Petri dish

Tefzel tubing (Bio-Rad)

Ultrasonic cleaner

Humidified chamber at 18-25ºC

METHOD

Coating Particles with dsRNA

1. Weigh 10 mg of the gold particles into a microfuge tube.

2. Add 100 µl of 0.05 M spermidine. Vortex the gold and spermidine mixture briefly. Sonicate for 3-5 seconds using an ultrasonic cleaner.

3. Add 20 µg of dsRNA dissolved in distilled H₂O to the spermidine-gold mixture. The volume of the dsRNA solution added should be less than 100 µl. Briefly vortex the dsRNA-spermidine-gold mixture at a moderate rate.

4. While vortexing the mixture, add 100 µl of 1 M CaCl₂ dropwise to the mixture. Make sure that the volume added is equal to that of the spermidine in Step 2.

5. Allow the mixture to precipitate for 10 minutes at room temperature.

6. Microfuge for 15 seconds and then remove the supernatant.

7. Wash the pellet three times with 1 ml of ethanol each time, vortex to disperse the pellet, and microfuge for 5 seconds to collect the pellet.

8. Add 100-200 µl of either ethanol or 0.05 mg/ml polyvinylpyrrolidone in ethanol (prepared fresh before use).

The final concentration of the coated gold suspension is 50-100 mg/ml. The choice of which concentration to use depends on whether 0.5 or 1 mg of gold per shot is delivered because each shot uses 10 µl of the suspension.

Polyvinylpyrrolidone serves as an adhesive and can increase the number of particles delivered at higher discharge pressures.

9. Use the suspension immediately in a gene gun or store up to 2 months at -20ºC.

Particle Bombardment with Gene Gun

1. Collect embryos 0-60 minutes after egg laying. Dechorionate the embryos with 50% bleach, wash with H₂O, and blot dry.

2. Use a fine-bristle paint brush to spread the embryos in a 20-mm-diameter lawn on a microslide or Petri dish that has been coated with either rubber glue or tape glue.

3. Desiccate the embryos for 5 minutes in the open air at room temperature. Bombard them with a single shot of gold-RNA.

i. Clamp the gun to a stationary aluminum rod frame. Orient the gun vertically such that the distance from nozzle to embryo plate below it is 2.5 cm.

CAUTION: Use hearing and eye protection for this procedure.

ii. Attach the gun to the flexible hose leading from the helium tank. The helium pressure regulator allows adjustment of pressure from 0 to 700 psi.

iii. Unlock the cartridge holder from the gun. Place on a flat surface with numbered edge facing up. Load an empty cartridge into position 1.

Cartridges are 0.5-inch pieces of Tefzel tubing (Bio-Rad) cut square with a razor blade.

iv. Vigorously mix the dsRNA-gold-ethanol slurry and pipette 10 µl of slurry into the tubing cartridge. This will contain 0.5-1.0 mg of gold particles and 1-2 µg of dsRNA.

v. Insert the loaded cartridge holder into the gene gun such that position 1 is in the firing position.

vi. Open the helium regulator and set the helium pressure to between 300 and 450 psi. Activate the safety interlock switch and press the trigger button to discharge the gold-dsRNA.

4. Immediately cover the embryos with a thin layer of halocarbon oil. Incubate in a humidified chamber at 18-25ºC until analysis.

ACKNOWLEDGMENTS

I am thankful to several people who generously provided their time and help during the preparation of this chapter. Young Sik Lee contributed information and illustrations. Jason Kennerdell and Shinji Yamaguchi provided extensive input into the design of many of these protocols and provided unpublished results. Mike Nonet provided information and advice for construction and operation of his gene gun. I thank members of my laboratory for helpful comments on the manuscript.