The Knockout Mouse Project

The goal of this project is to characterize mutant mice generated by the Knock-out Mouse Consortium (KOMP2) that have lethal phenotypes occurring before to E9.5. Thus far we have nearly 100 distinct KOMP2 knockout lines successfully pass through our phenotyping pipeline. We will provide date to the IMPC to be incorporated into the international effort to functionally annotate the mammalian genome. This data set will drastically increase the number of documented early phenotypes and we believe it is the largest consistent characterization of early phenotypes on record. 

This primary screen will provide baseline phenotypic data for many novel knockouts. To our knowledge this is the first systematic study of early lethal knockouts conducted using similar mutation strategies on the same genetic background (C57Bl/6JN). This large-scale analysis allows us to draw conclusions regarding developmental constraints during mammalian development and will provide a road map to early phenotypic characterization. 

Thus far we have found approximately equal distribution of phenotypes – approximately half gastrulation/perigastrulation phenotypes and the other half with preimplantation/implantation phenotypes. This is not unexpected as one of our original goals was to determine if there are specific milestones during development at which embryos would arrest or present phenotypic abnormalities (as opposed to a gradient of phenotypes across all time points).

Preimplantation Lethality Summary & Gastrulation Lethality Summary

Recent KOMP Publications

Regulation of Genomic Imprinting During Gastrulation

Genomic imprinting is an epigenetic phenomenon that results in parent of origin mono-allelic gene expression. The majority of imprinted genes reside in coordinately regulated clusters containing oppositely imprinted transcripts. Although allele specific DNA methylation and histone modifications have been shown at many imprinted loci, tissue and temporal specific mechanisms in vivo that establish and interpret these epigenetic marks are not fully defined. The mechanisms that regulate establishment and interpretation of epigenetic marks at imprinted loci are also essential for reprogramming – both during development in vivo and during induced pluripotency and somatic cell cloning.   Thus, understanding the full cadre of in vivo mechanisms regulating epigenetic events will guide our derivation and use of pluripotent cells.

We have discovered tissue specific regulation of genomic imprinting at the Igf2r locus during gastrulation in vivo. At the Igf2r/Airn imprinted cluster, paternal expression of the long non-coding RNA Airn has been shown to silence the paternal alleles of Igf2r, Slc22a2, and Slc22a3 resulting in maternal expression of these genes, and reciprocal imprinting at the locus. We have found that imprinting of Igf2r and Airn is regulated differently in distinct gastrulation lineages. Although imprinted expression is present in extraembryonic tissues at all stages, Igf2r is biallelic and Airn is not expressed in the epiblast prior to gastrulation. Once gastrulation commences, Igf2r and Airn become reciprocally imprinted. We also observe spreading of DNA methylation coincident with the start of gastrulation. Importantly, these results indicate epigenetic alterations occur as the epiblast differentiates into embryonic lineages, similar to what occurs as the zygotic genome is reprogrammed during preimplantation development.  We are continuing to define the mechanisms that are necessary and sufficient to achieve distinct lineage specific regulation of imprinting during gastrulation and determine the extent of epigenetic alterations in the newly formed germ layers using next-generation sequencing techniques.

Recent Publications

Marcho C, Cui W, Mager J. Epigenetic dynamics during preimplantation development. Reproduction. 2015;150(3):R109-R120. doi:10.1530/REP-15-0180