Dataset Information

TRIM21-mediated protein depletion recapitulates mutant gene phenotype in early mouse embryos

ABSTRACT: It is often desirable to be able to regulate or impair the expression of specific genes to study early developmental events in the mammalian embryo. While DNA and RNA methods are routine, methods using proteins are still in their infancy. When proteins in the cell encounter a specific antibody and the ubiquitin-protein ligase TRIM21, a ternary complex forms with the target protein, leading to its rapid and acute degradation – hence the name ‘Trim-Away'. However, there are many unknowns in this new endeavour. First and foremost, the extent to which endogenous proteins can be depleted depends on their amount in relation to the amount of exogenous antibody, which is limited by the microinjection procedure. Secondly, the depletion of the protein must be sustained over days. Using mass spectrometry and the iBAQ algorithm, we estimate the amount of proteins found in preimplantation mouse embryos. Most of these amounts are tractable with the microinjection method presented here, which supplies 10E-4 picomoles of antibody contained in 100 picolitres, before incurring toxic effects on mouse development. Building on these data, we demonstrate the feasibility of protein knock-down for a gene which is essential in the preimplantation mouse embryo, namely TEA domain family member 4 (Tead4). Knock-down persists long enough to result in a phenotype which is entirely consistent with that of the null mutation and the RNA interference: significantly reduced mRNA expression of TEAD4 target genes Cdx2 and Gata3, failure of CDX2 nuclear translocation and the embryo’s inability to implant. We conclude that at least for a time window of 3-4 days of preimplantation development, protein depletion is on the rise as a valid alternative to DNA and RNA methods.

INSTRUMENT(S): LTQ Orbitrap Velos, Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Embryo, Oocyte

SUBMITTER: Hannes Drexler

LAB HEAD: Hannes C. A. Drexler

PROVIDER: PXD012613 | Pride | 2019-09-11

REPOSITORIES: Pride

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			Action	DRS
	20110905_exp0313_HD_Michele_Oocyte-old-young_post-FASP_FT_01.raw	Raw
	20110905_exp0313_HD_Michele_Oocyte-old-young_post-FASP_pH11_02.raw	Raw
	20110905_exp0313_HD_Michele_Oocyte-old-young_post-FASP_pH3_07.raw	Raw
	20110905_exp0313_HD_Michele_Oocyte-old-young_post-FASP_pH4_06.raw	Raw
	20110905_exp0313_HD_Michele_Oocyte-old-young_post-FASP_pH5_05.raw	Raw

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Publications

A framework for TRIM21-mediated protein depletion in early mouse embryos: recapitulation of Tead4 null phenotype over three days.

Israel Steffen S Casser Ellen E Drexler Hannes C A HCA Fuellen Georg G Boiani Michele M

BMC genomics 20191021 1

<h4>Background</h4>While DNA and RNA methods are routine to disrupt the expression of specific genes, complete understanding of developmental processes requires also protein methods, because: oocytes and early embryos accumulate proteins and these are not directly affected by DNA and RNA methods. When proteins in the oocyte encounter a specific antibody and the TRIpartite Motiv-containing 21 (TRIM21) ubiquitin-protein ligase, they can be committed to degradation in the proteasome, producing a tr ...[more]

PMID: 31638890

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Project description:In order to study early developmental events in the mammalian embryo it is often desirable to be able to impair expression of specific genes. While DNA and RNA methods are routine, protein methods are still at the very beginning. When a specific antibody is supplied to mouse oocytes expressing the ubiquitin-protein ligase TRIM21, a ternary complex forms with the target protein, leading to its rapid and acute degradation - hence the name 'Trim-away' (PMID 29153837). However, there are many unknowns in this new endeavour. First and foremost, the extent to which endogenous proteins can be depleted depends on their amount, in relation to the amount of exogenous antibody, which is limited by the microinjection procedure. Secondly, the depletion of the protein must be sustained over days. Using the iBAQ algorithm we show that proteins found in preimplantation mouse embryos range from 3,5E-10 to 2,6E-02 picomoles. These amounts are tractable with our microinjection method, which supplies up to 100 picoliters and up to 6,7E-4 picomoles antibody before incurring in toxic effects on mouse development. Building on these data, we demonstrate the feasibility of protein knock-down for a gene which is essential in the preimplantation mouse embryo, namely TEAD4 (TEA domain family member 4). Protein knock-down persists for sufficient time to result in a phenotype which is entirely consistent with that of the null mutation (Tead4 -/-) and of the RNA interference, namely: significantly reduced mRNA expression of TEAD4 target genes Cdx2 and Gata3, and embryo’s inability to implant. We conclude that for a time window of 3-4 days of preimplantation development the protein depletion method can be a valid alternative to DNA and RNA methods. After in vivo fertilization and short culture in KSOM(aa) medium, pronuclear-stage oocytes (B6C3F1 x CD1) were microinjected with Trim21 mRNA and dextran beads as tracer (named 'group 4'), or Trim21 mRNA, dextran beads and anti-GFP antibody (named 'group 5'), or Trim21 mRNA, dextran beads and anti-TEAD4 antibody (named 'group 6'), two replicates each ('a' and 'b'). On day 4 after microinjection, the most advanced embryos were examined for phenotype or lysed for transcriptome analysis. TEAD4-depleted embryos formed only 30% blastocysts and these were not able to implant in the outgrowth assay, in contrast to the almost full rates of the embryos injected with Trim21 mRNA or Trim21 mRNA + anti-GFP antibody. Transcriptome analysis revealed that the TEAD4 target genes Cdx2 and Gata3 are significantly reduced in the embryos that received the anti-TEAD4 antibody compared to the embryos that received Trim21 mRNA only, while the embryos that received anti-GFP antibody were much similar to those that received Trim21 mRNA only.