Quantitative Microinjection of Morpholino Antisense Oligonucleotides into Mouse Oocytes to Examine Gene Function in Meiosis-I
Specific protein depletion is a powerful approach for assessing individual gene function in cellular processes, and has been extensively employed in recent years in mammalian oocyte meiosis-I. Conditional knockout mice and RNA interference (RNAi) methods such as siRNA or dsRNA microinjection are among several approaches to have been applied in this system over the past decade. RNAi by microinjection of Morpholino antisense Oligonucleotides (MO), in particular, has proven highly popular and tractable in many studies, since MOs have high specificity of interaction, low cell toxicity, and are more stable than other microinjected RNAi molecules. Here, we describe a method of MO microinjection into the mouse germinal vesicle-stage (GV) oocyte followed by a simple immunofluorescence approach for examination of gene function in meiosis-I.
Key wordsMorpholino antisense oligonucleotides Microinjection Mouse oocyte Meiosis-I Immunofluorescence
Works in GFs lab is supported by CIHR, NSERC, CFI, and Fondation Jean-Louis Lévesque. Elements of the described procedure were learned from labs of John Carroll, Karl Swann, Jay Baltz, and Tomohiro Kono. We thank Jenna Haverfield, Cayetana Vázquez-Diez, and Angus MacCaulay for critical reading of the manuscript.