Protocol
Microinjection of dsRNA into Fully Grown Mouse Oocytes Paula Stein Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA
INTRODUCTION RNA interference (RNAi) is a suitable method for sequence-specific post-transcriptional gene silencing in a number of model systems. The following protocol describes delivery of a double-stranded RNA (dsRNA) of choice into fully grown, germinal vesicle-intact (GV) mouse oocytes by microinjection. Microinjected oocytes can be cultured for up to 2 d or they can be matured to metaphase II. The metaphase II eggs can be fertilized in vitro and cultured up to the blastocyst stage. The efficiency of knockdown by RNAi can be assayed by quantitative reverse transcriptase (RT)-PCR (qPCR).
RELATED INFORMATION This protocol is an updated version of Microinjection of dsRNA into Mouse Oocytes and Early Embryos (Stein and Svoboda 2006). Protocols for Preparation of dsRNA for Microinjection Experiments in Mouse (Svoboda 2009) and Making Injection Pipettes (Nagy et al. 2006) are also available. Background information is provided in RNAi Experiments in Mouse Oocytes and Early Embryos (Svoboda and Stein 2009).
MATERIALS CAUTIONS AND RECIPES: Please see Appendices for appropriate handling of materials marked with , and recipes for reagents marked with .
Reagents CZB medium containing 7 mM taurine (CZBT)
dsRNA solution (≥106 molecules in 10 pL) IBMX (3-isobutyl-1-methylxanthine) (0.2 M stock solution in DMSO) See Step 1 for other reagents which can be used in place of IBMX.
Injection medium MEM/PVP containing 0.2 mM IBMX is typically used. Whitten’s/HEPES/PVA can be used as an alternative. MEM/PVP medium Mice, NSA (CF-1) (6-wk old females) (Harlan) Fully grown, germinal vesicle (GV)-intact mouse oocytes are obtained from these mice. To improve the yield of antral follicles containing fully grown oocytes, give mice a single injection of 5 IU of pregnant mare’s serum gonadotropin (PMSG) intraperitoneally 48 h prior to sacrifice.
Corresponding author (
[email protected]) Cite as: Cold Spring Harb. Protoc.; 2009; doi:10.1101/pdb.prot5132 © 2009 Cold Spring Harbor Laboratory Press
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Equipment Dishes, tissue culture (sterile plastic) (35-, 60-, and 100-mm) Instead of a 100-mm Petri dish, a Nunc Lab-Tek chamber slide can be used to accommodate the microinjection drops (Step 6).
Forceps Incubator (humidified; 5% CO2 in air at 37ºC) Microinjection setup (micromanipulator and injector [e.g. Picoinjector; Harvard Apparatus and Femtojet; Eppendorf]) Microscope Needle (27-gauge) Paraffin oil (light) Pipettes, glass (mouth-operated) Pipettes, injection and holding Make injection pipettes by pulling borosilicate-glass capillary tubing in a mechanical pipette puller. They can be prepared in advance or as microinjection proceeds. Pull holding pipettes the same way, but cut them to a diameter of 80-120 µm and melt the tip using a microforge. Prepare them in advance (for detailed instructions, see Making Injection Pipettes [Nagy et al. 2006]). Holding pipettes are commercially available from various companies, e.g., Eppendorf, Humagen, etc.
Scissors Syringe (1-mL) Watch glasses or glass staining blocks (VWR)
METHOD A typical experimental outline of an RNAi experiment in mouse oocytes is shown in Figure 1. Specific protocols and information on preparation for experiments in mouse oocytes and early embryos are provided in the text by Nagy et al. (2003).
FIGURE 1. An outline of RNAi experiments in mouse oocytes. Culture conditions are shown on the left. (CZB: culture medium; IBMX, 3-isobutyl-1-methylxanthine [inhibitor of meiotic resumption]). Microinjected oocytes are cultured in IBMXcontaining medium, typically for 20 h. This period of culture extends the exposure of oocytes to the dsRNA and, especially in the case of dormant maternal messages, allows for mRNA degradation to occur prior to appearance of the protein after resumption of meiosis. www.cshprotocols.org
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Collection of Mouse Oocytes The time required for this portion of the method is ~1 h. 1. Prepare the culture medium (CZBT+IBMX): Add 2 µL of 0.2 M IBMX to 2 mL of CZBT (Chatot et
al. 1989). IMPORTANT: The removal of oocytes from the ovarian follicles results in spontaneous resumption of meiosis. To prevent this and keep the oocytes at the GV stage, the levels of cAMP must be kept high in all collection, microinjection, and culture media used for oocytes. Several reagents can be added for this purpose: IBMX, a phosphodiesterase (PDE) inhibitor, milrinone, an inhibitor of PDE3, or cAMP analogs (typically dibutyryl cAMP). 2. Set up microdrop culture dishes: Place several 75-µL drops of CZBT+IBMX on the bottom of a 60-
mm sterile plastic tissue culture dish and cover the dish with light paraffin oil. Place the dish in a humidified incubator containing 5% CO2 in air at 37ºC. Culture dishes should be set up in advance to allow for temperature and CO2 equilibration. 3. Sacrifice the females by cervical dislocation and remove the ovaries. Place the ovaries in a watch
glass with MEM/PVP containing 0.2 mM IBMX. 4. Release the antral follicles from the ovaries by puncturing them several times with a 27-gauge nee-
dle attached to a 1-mL syringe. Use a mouth-operated glass pipette to collect the oocyte-cumulus cell complexes and transfer them to a clean watch glass containing 1-3 mL of MEM/PVP+IBMX. Select large antral follicles containing cumulus-enclosed oocytes and avoid collecting the smaller pre-antral follicles or denuded oocytes. 5. Use a pipette whose tip diameter is about the size of the oocytes to pipette the complexes up and
down to detach the cumulus cells. Transfer the cumulus-free oocytes to the culture dish and place in the incubator. After collection, keep oocytes in culture for at least 30 min to allow recovery before microinjection.
Microinjection of Mouse Oocytes The time required for this portion of the method is ~2 h. 6. Set up the micromanipulator for injection. i.
Place a 5-µL drop of injection medium (microinjection drop) (e.g., MEM/PVP+IBMX) 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. When more than one dsRNA is injected, or if vehicle injection (usually H2O) is used as a control, prepare a new set of drops (i.e., a 5 µL-drop of MEM/PVP+IBMX and a 1 µL-drop of dsRNA or H2O) for each substance to be microinjected. iii. Place the dish on the stage of the micromanipulator, position the injection and holding
pipettes, and connect the gas supply. 7. Transfer a group of oocytes 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 are microinjected. Check all microinjected cells under the microscope and remove those that did not survive. See Troubleshooting.
Culture and Maturation of Mouse Oocytes The time required for this portion of the method is 1-2 d. 8. Culture the oocytes in CZBT+IBMX for 20-24 h. Cells can then be lysed and assayed by qPCR; see Discussion. See Troubleshooting. 9. If meiotic maturation is required, wash the oocytes with several drops of IBMX-free CZBT and cul-
ture them in CZBT for 16-18 h.
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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. The longer the culture period for oocytes denuded of the granulosa cells, the worse developmental potential the oocytes have. Thus, culturing conditions should be highly optimized and culture periods should be as short as possible to minimize negative effects. Alternatively, using granulosa cell-enclosed oocytes for microinjection can help when longer culturing periods are needed. This approach is more difficult because of debris accumulation on the injection pipette, but it has a very positive effect on quality of the cultured oocytes.
TROUBLESHOOTING Problem: Oocytes exhibit low survival rates after microinjection. [Step 7] Solution: Consider the following: 1. The volume injected may be too large. Prepare injection pipettes with a smaller diameter tip. 2. The microinjection may have taken too long. Transfer fewer oocytes to the micromanipulator; the
less time the cells stay outside of the incubator, the better their rate of survival. 3. Something in the dsRNA solution may be toxic to the oocytes. Precipitate the dsRNA again, wash
with 75% ethanol, air-dry, and resuspend in H2O. Problem: No RNAi effect is observed in the injected oocytes. [Step 8] Solution: The dsRNA may not be effective. Try a more concentrated solution of dsRNA or make a
longer dsRNA molecule. Alternatively, try longer culture times.
DISCUSSION The duration of a microinjection experiment depends on the phenotypic characteristic studied. Cell collection and microinjection of dsRNA are completed in 1 d but, in most experiments, the GV oocytes are cultured for 1-2 d before they are assayed, and for another 16-18 h if the experiment requires maturing the GV oocytes to metaphase II eggs. If necessary, the matured eggs can be fertilized in vitro and further cultured to assess embryo development. This adds anywhere from 1 to 4 d, depending on what stage of development needs to be reached. In addition, female mice must be primed with gonadotropins 48 h prior to oocyte collection. The first assay conducted with microinjected oocytes should always be qPCR to check that the cognate mRNA has been degraded, i.e., to prove that the dsRNA is producing an RNAi effect. Specific biological assays can range from assessing the development or morphology of the cells under the microscope to techniques such as Western blot, immunofluorescence, or enzymatic activity determination.
REFERENCES Chatot, C.L., Ziomek, C.A., Bavister, B.D., Lewis, J.L., and Torres, I. 1989. An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro. J. Reprod. Fertil. 86: 679–688. Nagy, A., Gertsenstein, M., Vintersten, K., and Behringer, R. 2003. Manipulating the mouse embryo: A laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Nagy, A., Gertsenstein, M., Vintersten, K., and Behringer, R. 2006. Making injection pipettes. Cold Spring Harb. Protoc. doi: 10.1101/ pdb.prot4395.
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Stein, P. and Svoboda, P. 2006. Microinjection of dsRNA into mouse oocytes and early embryos. Cold Spring Harb. Protoc. doi: 10.1101/pdb.prot4511. Svoboda, P. 2009. Preparation of dsRNA for microinjection experiments in mouse. Cold Spring Harb. Protoc. (this issue). doi: 10.1101/pdb.prot5131. Svoboda, P. and Stein, P. 2009. RNAi experiments in mouse oocytes and early embryos. Cold Spring Harb. Protoc. (this issue). doi: 10.1101/pdb.top56.
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