Collection of Early Mouse Embryos for RNAi

Stein, Paxton

doi:10.1101/pdb.prot4510

Cited by 2 publications

(1 citation statement)

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“…Fully grown, germinal vesicle (GV)-intact oocytes, metaphase II (MII) eggs, and 1-cell and 2-cell embryos were collected as previously described [41,42]. GV oocytes were cultured in Chatot-Ziomek-Bavister (CZB) medium containing 0.2 mM isobutylmethylxanthine (IBMX; Sigma) to inhibit GV breakdown.…”

Section: Mouse Oocyte/egg/embryo Collection Cell Culture and Microimentioning

confidence: 99%

Lin28a Is Dormant, Functional, and Dispensable During Mouse Oocyte-to-Embryo Transition1

Flemr

Moravec

Libova

et al. 2014

Biology of Reproduction

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The oocyte-to-embryo transition (OET) denotes transformation of a highly differentiated oocyte into totipotent blastomeres of the early mammalian embryo. OET depends exclusively on maternal RNAs and proteins accumulated during oocyte growth, which implies importance of post-transcriptional control of gene expression. OET includes replacement of abundant maternal microRNAs (miRNAs), enriched also in differentiated cells and exemplified by the Let-7 family, with embryonic miRNAs common in pluripotent stem cells (the miR-290 family in the mouse). Lin28a and its homolog Lin28b encode RNA-binding proteins, which interfere with Let-7 maturation and facilitate reprogramming of induced pluripotent stem cells. Both Lin28a and Lin28b transcripts are abundant in mouse oocytes. To test the role of maternal expression of Lin28a and Lin28b during oocyte-to-zygote reprogramming, we generated mice with oocyte-specific knockdown of both genes by using transgenic RNA interference. Lin28a and Lin28b are dispensable during oocyte growth because their knockdown has no effect on Let-7a levels in fully grown germinal vesicle (GV)-intact oocytes. Furthermore, transgenic females were fertile and produced healthy offspring, and their overall breeding performance was comparable to that of wild-type mice. At the same time, 2-cell embryos derived from transgenic females showed up-regulation of mature Let-7, suggesting that maternally provided LIN28A and LIN28B function during zygotic genome activation. Consistent with this conclusion is increased translation of Lin28a transcripts upon resumption of meiosis. Our data imply dual repression of Let-7 during OET in the mouse model, the selective suppression of Let-7 biogenesis by Lin28 homologs superimposed on previously reported global suppression of miRNA activity.

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Section: Mouse Oocyte/egg/embryo Collection Cell Culture and Microimentioning

confidence: 99%

Lin28a Is Dormant, Functional, and Dispensable During Mouse Oocyte-to-Embryo Transition1

Flemr

Moravec

Libova

et al. 2014

Biology of Reproduction

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RNAi Experiments in Mouse Oocytes and Early Embryos

Svoboda¹,

Stein²

2009

Cold Spring Harb Protoc

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The discovery of RNA interference (RNAi) in 1998 ushered in a new era in biology. RNAi currently serves as a favorite approach for inhibition of gene function in many areas of research. This article provides a brief review of RNAi and discussion of the benefits and drawbacks of using long double-stranded RNA (dsRNA) in mammalian oocytes and early embryos. We also provide an introduction to protocols for RNAi experiments in mouse, including preparation and microinjection of dsRNA into mouse oocytes and early embryos, and preparation and testing of constructs for transgenic RNAi based on long hairpin RNA expression.

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Collection of Early Mouse Embryos for RNAi

Cited by 2 publications

References 0 publications

Lin28a Is Dormant, Functional, and Dispensable During Mouse Oocyte-to-Embryo Transition1

Lin28a Is Dormant, Functional, and Dispensable During Mouse Oocyte-to-Embryo Transition1

RNAi Experiments in Mouse Oocytes and Early Embryos

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