Developmental Biology Mammalia Genetics and Genomics

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Authors: Paula Stein and Petr Svoboda

This protocol was adapted from “RNAi in Mouse Oocytes and Early Embryos” contributed by Paula Stein and Petr Svoboda, Chapter 15, in RNAi: A Guide to Gene Silencing (ed. Hannon). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

INTRODUCTION

This protocol describes how to introduce a double-stranded RNA (dsRNA) of choice into mouse oocytes or fertilized one-cell embryos by microinjection. For collection of mouse oocytes and early embryos, see Collection of Mouse Oocytes for RNAi and Collection of Early Mouse Embryos for RNAi. It is advisable to confirm knock-down of the gene of interest by RT-PCR (see RNA Isolation and RT-PCR from dsRNA-treated Mouse Oocytes and Early Embryos).

MATERIALS

Reagents

  1. CZB medium
  2. dsRNA solution (see Preparation of dsRNA Molecules for RNAi in Mouse Oocytes and Early Embryos)
  3. DMSO containing 0.2M IBMX (3-isobutyl-1-methylxanthine)
  4. Injection media: MEM/PVP
    • MEM/PVP is for use with embryos. For oocytes, use MEM/PVP containing 0.2 mM IBMX
  5. KSOM medium
  6. Oocytes or embryos (see Collection of Mouse Oocytes for RNAi and Collection of Early Mouse Embryos for RNAi)
    • After collection, oocytes and embryos should be kept in culture for at least 30 minutes to allow recovery before microinjection.

Equipment

  1. Gas supply
  2. Incubator
  3. Injection and holding pipettes
    • Injection pipettes are made by pulling borosilicate-glass capillary tubing on a mechanical pipette puller. They can be prepared in advance or as microinjection proceeds. Holding pipettes are pulled the same way, but they must be cut to a diameter of 80-120 μm and the tip melted using a microforge. They are prepared in advance.
  4. Micromanipulator
  5. Paraffin oil (light)
  6. Tissue culture dishes (sterile plastic 100-mm)

METHOD

  1. Set up the micromanipulator for injection.
    • i. Place a 5-μl drop of injection medium (microinjection drop) on the top of a 100-mm plastic tissue culture dish.
    • ii. Place a 1-μl drop of dsRNA solution as close as possible to the other drop and then flood the dish with light paraffin oil.
    • iii. Place the dish in the stage of the micromanipulator, position the injection and holding pipettes, and connect the gas supply.
      • When more than one dsRNA is injected or vehicle (usually H2O) injection is used as a control, set up a new set of drops (a 5 μl-drop of MEM/PVP [+IBMX] and a 1 μl-drop of dsRNA or H2O) for each substance to be microinjected.
  2. Transfer a group of oocytes/embryos from the incubator to the microinjection drop and inject 5-10 pl of dsRNA into their cytoplasm. Place them back in the incubator. Repeat the procedure with another group until all oocytes/embryos are microinjected. Check all microinjected cells under the microscope and remove those that did not survive.
  3. Culture the oocytes or early embryos.
    • i. Culture the oocytes in CZB containing 0.2 mM IBMX for 20-24 hours. They can then be lysed and assayed by RT-PCR (see RNA Isolation and RT-PCR from dsRNA-treated Mouse Oocytes and Early Embryos). If meiotic maturation is required, wash the oocytes with several drops of IBMX-free CZB and culture them in CZB for 16-18 hours. The in-vitro-matured metaphase II eggs can be processed for immunofluorescence, western blot, or enzyme activity measurements, or they can be fertilized in vitro.
    • ii. Culture one-cell embryos in KSOM up to the desired stage.
      • If one-cell embryos will be cultured only up to the two-cell stage, the culture can be done in CZB. For longer cultures, however, KSOM is preferred, as it enhances embryo development to later stages. In addition, the development is improved when the culture is performed in at atmosphere of 5% CO2/5% O2/90% N2.

Troubleshooting

  1. Problem: Low survival after microinjection. [Step 3]
    • Solution: (1) Possibly the volume injected was too large. Prepare injection pipettes with a smaller diameter opening. (2) Something in the dsRNA solution is toxic to the cells. Precipitate the dsRNA again, wash it with 75% ethanol, air-dry, and resuspend it in H2O. (3) The microinjection may have taken too long. Transfer fewer oocytes/embryos to the micromanipulator. The less time the cells spend outside the incubator, the better their survival and development.
  2. Problem: Poor in vitro development of the embryos. [Step 3]
    • Solution: Possibly, the embryos were outside the incubator for too long. Transfer fewer embryos to the micromanipulator, inject them quickly, and expose them to as little light as possible.
  3. Problem: No RNAi effect.
    • Solution: Possibly the dsRNA is not effective. Try a more concentrated solution of dsRNA or make a longer dsRNA molecule. Alternatively, try longer culture times.

ACKNOWLEDGMENTS

The authors thank Dr. Richard M. Schultz, in whose lab all of the research and development of protocols presented in this work were conducted and supported by a grant from the National Institutes of Health (HD 22681). Previous Section

References

  1. Nagy A., Gertsenstein M., Vintersten K., and Behringer R. 2003. Manipulating the mouse embryo: A laboratory manual, 3rd. Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

DOI

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