Mammalian blastocysts comprise three distinct lineages, namely, trophoblast, hypoblast, and epiblast, which develop into fetal placenta, extraembryonic yolk sac, and embryo proper, respectively. Pluripotent embryonic stem cells, capable of forming all adult cell types, can only be derived from the epiblast. In mouse and rat, this process is promoted by the double inhibition (2i) of mitogen-activated protein kinase kinase (MAP2K), which antagonizes FGF signaling, and glycogen synthase kinase 3 (GSK3), which stimulates the WNT pathway. We investigated variations of the 2i treatment on lineage segregation and pluripotency-related gene expression in bovine blastocysts. In vitro-fertilized embryos were cultured either in the presence of inhibitors of GSK3 (3 μM CHIR) and MAP2K (0.4 vs. 10 μM PD0325901, designated 2i and 2i+, respectively) or in 2i/2i+ with FGFR inhibitor (0.1 μM PD173074, designated 3i [2i and PD173074] and 3i+ [2i+ and PD173074]). Compared with 2i, both 2i+ and 3i+ potentiated the improvement in blastocyst morphology. Using an automated platform for multiplexed digital mRNA profiling, we simultaneously counted transcripts of 76 candidate genes in bovine blastocysts treated with multiple kinase inhibitors. We show that 2i+ medium specifically increased FGF4 and NANOG while reducing PDGFRalpha and SOX17 levels. The shift from a hypoblast to an epiblast gene expression signature was confirmed by quantitative PCR. A wide range of functionally related genes, including candidates involved in DNA methylation, were not significantly changed. This well-defined 2i+ effect was not observed after pharmacologically inhibiting FGF receptor or related MAP kinases (p38, JNK, and ERK5). In summary, our data suggest that increased MAP2K inhibition exerts its pluripotency-promoting effects through as yet unidentified signals.

No abstract

Selecting beneficial DNA variants is the main goal of animal breeding. However, this process is inherently inefficient because each animal only carries a fraction of all desirable variants. Genome editing technology with its ability to directly introduce beneficial sequence variants offers new opportunities to modernize animal breeding by overcoming this biological limitation and accelerating genetic gains. To realize rapid genetic gain, precise edits need to be introduced into genomicallyselected embryos, which minimizes the genetic lag. However, embryo-mediated precision editing by homology-directed repair (HDR) mechanisms is currently an inefficient process that often produces mosaic embryos and greatly limits the numbers of available edited embryos. This review provides a summary of genome editing in bovine embryos and proposes an embryo-mediated accelerated breeding scheme that overcomes the present efficiency limitations of HDR editing in bovine embryos. It integrates embryo-based genomic selection with precise multi-editing and uses embryonic cloning with elite edited blastomeres or embryonic pluripotent stem cells to resolve mosaicism, enable multiplex editing and multiply rare elite genotypes. Such a breeding strategy would enable a more targeted, accelerated approach for livestock improvement that allows stacking of beneficial variants, even including novel traits from outside the breeding population, in the most recent elite genetic background, essentially within a single generation.

Genome-wide association studies (GWAS) of Huntington’s disease (HD) have identified six DNA maintenance gene loci (among others) as modifiers and implicated a two step-mechanism of pathogenesis: somatic instability of the causative HTT CAG repeat with subsequent triggering of neuronal damage. The largest studies have been limited to HD individuals with a rater-estimated age at motor onset. To capitalize on the wealth of phenotypic data in several large HD natural history studies, we have performed algorithmic prediction using common motor and cognitive measures to predict age at other disease landmarks as additional phenotypes for GWAS. Combined with imputation using the Trans-Omics for Precision Medicine reference panel, predictions using integrated measures provided objective landmark phenotypes with greater power to detect most modifier loci. Importantly, substantial differences in the relative modifier signal across loci, highlighted by comparing common modifiers at MSH3 and FAN1, revealed that individual modifier effects can act preferentially in the motor or cognitive domains. Individual components of the DNA maintenance modifier mechanisms may therefore act differentially on the neuronal circuits underlying the corresponding clinical measures. In addition, we identified new modifier effects at the PMS1 and PMS2 loci and implicated a potential new locus on chromosome 7. These findings indicate that broadened discovery and characterization of HD genetic modifiers based on additional quantitative or qualitative phenotypes offers not only the promise of in-human validated therapeutic targets, but also a route to dissecting the mechanisms and cell types involved in both the somatic instability and toxicity components of HD pathogenesis.

Zona-free somatic cell transfer (SCT) and embryo aggregation increase throughput and efficiency of cloned embryo and offspring production, respectively, but both approaches have not been widely adopted. Cloning efficiency is further improved by cell cycle coordination between the interphase donor cell and metaphasearrested recipient cytoplast. This commonly involves inclusion of caffeine and omission of calcium to maintain high mitotic cyclin-dependent kinase activity and low calcium levels, respectively, in the nonactivated cytoplast. The aim of our study was to integrate these various methodological improvements into a single work stream that increases sheep cloning success. We show that omitting calcium during zona-free SCT improved blastocyst development from 6% to 13%, while caffeine treatment reduced spontaneous oocyte activation from 17% to 8%. In a retrospective analysis, morula aggregation produced high morphological quality blastocysts with better in vivo survival to term than nonaggregated controls (15% vs. 9%), particularly after vitrification (14% vs. 0%). By combining cytoplast cell cycle control with zona-free embryo reconstruction and aggregation, this novel SCT protocol maximizes the benefits of vitrification by producing more cryoresilient blastocysts. The presented cloning methodology is relatively easy to operate and further increases throughput and efficiency of cloned embryo and offspring production. Integration of additional reprogramming steps or alternate donor cells is straightforward, providing a flexible workflow that can be adapted to changing experimental requirements.

Multiplying the germline would increase the number of offspring that can be produced from selected animals, accelerating genetic improvement for livestock breeding. This could be achieved by producing multiple chimaeric animals, each carrying a mix of donor and host germ cells in their gonads. However, such chimaeric germlines would produce offspring from both donor and host genotypes, limiting the rate of genetic improvement. To resolve this problem, we disrupted the RNA‐binding protein DAZL and generated germ cell‐deficient host animals. Using Cas9‐mediated homology‐directed repair (HDR), we introduced a DAZL loss‐of‐function mutation in male ovine fetal fibroblasts. Following manual single cell isolation, 4/48 (8.3%) of donor cell strains were homozygously HDR‐edited. Sequence‐validated strains were used as nuclear donors for somatic cell cloning to generate three lambs, which died at birth. All DAZL null male neonatal sheep lacked germ cells on histological sections and showed greatly reduced germ cell markers. Somatic cells within their testes were morphologically intact and expressed normal levels of lineage‐specific markers, suggesting that the germ cell niche remained intact. This extends the DAZL mutant phenotype beyond mice into agriculturally relevant ruminants, providing a pathway for using absolute germline transmitters in rapid livestock improvement.

Multiplying the germline would increase the number of offspring that can be produced from selected animals, accelerating genetic improvement for livestock breeding. This could be achieved by producing multiple chimaeric animals, each carrying a mix of donor and host germ cells in their gonads. However, such chimaeric germlines would produce offspring from both donor and host genotypes, limiting the rate of genetic improvement. To resolve this problem and produce chimaeras with absolute donor germline transmission, we have disrupted the RNA-binding protein DAZL and generated germ cell-deficient host animals.Using Cas9 mediated homology-directed repair (HDR), we introduced a DAZL loss-offunction mutation in male ovine fetal fibroblasts. Following manual single-cell isolation, 4/48 (8.3%) of donor cell strains were homozygously HDR-edited. Sequence-validated strains were used as nuclear donors for somatic cell cloning to generate three lambs, which died at birth. All DAZL-null male neonatal sheep lacked germ cells. Somatic cells within their testes were morphologically intact and expressed normal levels of somatic cell-specific marker genes, indicating that the germ cell niche remained intact. This extends the DAZL-mutant phenotype beyond mice into agriculturally relevant ruminants, providing a pathway for using absolute transmitters in rapid livestock improvement.

This protocol summarises zona-free sheep cloning by somatic cell transfer (SCT), adapted from a similar SCT method in cattle (