Genome edited sheep and cattle.

Genome editing tools enable efficient and accurate genome manipulation. An enhanced ability to modify the genomes of livestock species could be utilized to improve disease resistance, productivity or breeding capability as well as the generation of new biomedical models. To date, with respect to the direct injection of genome editor mRNA into livestock zygotes, this technology has been limited to the generation of pigs with edited genomes. To capture the far-reaching applications of gene-editing, from disease modelling to agricultural improvement, the technology must be easily applied to a number of species using a variety of approaches. In this study, we demonstrate zygote injection of TALEN mRNA can also produce gene-edited cattle and sheep. In both species we have targeted the myostatin (MSTN) gene. In addition, we report a critical innovation for application of gene-editing to the cattle industry whereby gene-edited calves can be produced with specified genetics by ovum pickup, in vitro fertilization and zygote microinjection (OPU-IVF-ZM). This provides a practical alternative to somatic cell nuclear transfer for gene knockout or introgression of desirable alleles into a target breed/genetic line.

Maternal nutrient restriction in late pregnancy programs postnatal metabolism and pituitary development in beef heifers.

Maternal undernutrition during pregnancy followed by ad libitum access to nutrients during postnatal life induces postnatal metabolic disruptions in multiple species. Therefore, an experiment was conducted to evaluate postnatal growth, metabolism, and development of beef heifers exposed to late gestation maternal nutrient restriction. Pregnancies were generated via transfer of in vitro embryos produced using X-bearing sperm from a single Angus sire. Pregnant dams were randomly assigned to receive either 100% (control; n = 9) or 70% (restricted; n = 9) of their total energy requirements from gestational day 158 to parturition. From post-natal day (PND) 301 until slaughter (PND485), heifers were individually fed ad libitum in a Calan gate facility. Calves from restricted dams were lighter than controls at birth (P<0.05) through PND70 (P<0.05) with no difference in body weight from PND105 through PND485 (P>0.10). To assess pancreatic function, glucose tolerance tests were performed on PND315 and PND482 and a diet effect was seen with glucose area under the curve being greater (P<0.05) in calves born to restricted dams compared to controls. At slaughter, total internal fat was greater (P<0.05) in heifers born to restricted dams, while whole pituitary weight was lighter (P<0.05). Heifers from restricted dams had fewer growth hormone-positive cells (somatotrophs) compared to controls (P<0.05). Results demonstrate an impaired ability to clear peripheral glucose in heifers born to restricted dams leading to increased deposition of internal fat. A reduction in the number of somatotrophs may contribute to the adipogenic phenotype of heifers born to restricted dams due to growth hormone's known anabolic roles in growth, lipolysis, and pancreatic islet function.

Oxygen-induced alterations in the expression of chromatin modifying enzymes and the transcriptional regulation of imprinted genes.

Embryo culture and assisted reproductive technologies have been associated with a disproportionately high number of epigenetic abnormalities in the resulting offspring. However, the mechanisms by which these techniques influence the epigenome remain poorly defined. In this study, we evaluated the capacity of oxygen concentration to influence the transcriptional control of a selection of key enzymes regulating chromatin structure. In mouse embryonic stem cells, oxygen concentrations modulated the transcriptional regulation of the TET family of enzymes, as well as the de novo methyltransferase Dnmt3a. These transcriptional changes were associated with alterations in the control of multiple imprinted genes, including H19, Igf2, Igf2r, and Peg3. Similarly, exposure of in vitro produced bovine embryos to atmospheric oxygen concentrations was associated with disruptions in the transcriptional regulation of TET1, TET3, and DNMT3a, along with the DNA methyltransferase co-factor HELLS. In addition, exposure to high oxygen was associated with alterations in the abundance of transcripts encoding members of the Polycomb repressor complex (EED and EZH2), the histone methyltransferase SETDB1 and multiple histone demethylases (KDM1A, KDM4B, and KDM4C). These disruptions were accompanied by a reduction in embryo viability and suppression of the pluripotency genes NANOG and SOX2. These experiments demonstrate that oxygen has the capacity to modulate the transcriptional control of chromatin modifying genes involved in the establishment and maintenance of both pluripotency and genomic imprinting.

Genetic engineering a large animal model of human hypophosphatasia in sheep.

The availability of tools to accurately replicate the clinical phenotype of rare human diseases is a key step toward improved understanding of disease progression and the development of more effective therapeutics. We successfully generated the first large animal model of a rare human bone disease, hypophosphatasia (HPP) using CRISPR/Cas9 to introduce a single point mutation in the tissue nonspecific alkaline phosphatase (TNSALP) gene (ALPL) (1077 C > G) in sheep. HPP is a rare inherited disorder of mineral metabolism that affects bone and tooth development, and is associated with muscle weakness. Compared to wild-type (WT) controls, HPP sheep have reduced serum alkaline phosphatase activity, decreased tail vertebral bone size, and metaphyseal flaring, consistent with the mineralization deficits observed in human HPP patients. Computed tomography revealed short roots and thin dentin in incisors, and reduced mandibular bone in HPP vs. WT sheep, accurately replicating odonto-HPP. Skeletal muscle biopsies revealed aberrant fiber size and disorganized mitochondrial cristae structure in HPP vs. WT sheep. These genetically engineered sheep accurately phenocopy human HPP and provide a novel large animal platform for the longitudinal study of HPP progression, as well as other rare human bone diseases.

Histone-lysine N-methyltransferase SETDB1 is required for development of the bovine blastocyst.

Transcripts derived from select clades of transposable elements are among the first to appear in early mouse and human embryos, indicating transposable elements and the mechanisms that regulate their activity are fundamental to the establishment of the founding mammalian lineages. However, the mechanisms by which these parasitic sequences are involved in directing the developmental program are still poorly characterized. Transposable elements are regulated through epigenetic means, where combinatorial patterns of DNA methylation and histone 3 lysine 9 trimethylation (H3K9me3) suppress their transcription. From studies in rodents, SET domain bifurcated 1 (SETDB1) has emerged as the core methyltransferase responsible for marking transposable elements with H3K9me3 and temporally regulating their transcriptional activity. SETDB1 loss of function studies in mice reveal that although extraembryonic tissues do not require this methyltransferase, establishment of the embryo proper fails without it. As the bovine embryo initiates the processes of epigenetic programming earlier in the preimplantation phase, we sought to determine whether suppressing SETDB1 would block the formation of the inner cell mass. We report here that bovine SETDB1 transcripts are present throughout preimplantation development, and RNA interference-based depletion blocks embryo growth at the morula stage of development. Although we did not observe alterations in global histone methylation or transposable element transcription, we did observe increased global levels of H3K27 acetylation, an epigenetic mark associated with active enhancers. Our observations suggest that SETDB1 might interact with the epigenetic machinery controlling enhancer function and that suppression of this methyltransferase may disrupt the bovine developmental program.