Genome editing tools have revolutionized the generation of genetically modified animals including livestock. In particular, the domestic pig is a proven model of human physiology and an agriculturally important species. In this study, we utilized the CRISPR/Cas9 system to edit the NANOS2 gene in pig embryos to generate offspring with mono-allelic and bi-allelic mutations. We found that NANOS2 knockout pigs phenocopy knockout mice with male specific germline ablation but other aspects of testicular development are normal. Moreover, male pigs with one intact NANOS2 allele and female knockout pigs are fertile. From an agriculture perspective, NANOS2 knockout male pigs are expected to serve as an ideal surrogate for transplantation of donor spermatogonial stem cells to expand the availability of gametes from genetically desirable sires.

The domestic pig is an important “dual purpose” animal model for agricultural and biomedical applications. There is an emerging consensus in the biomedical community for the use of large animal models such as pigs to either serve as an alternative, or complement investigations from the mouse. However, the use of pig has not proven popular due to technical difficulties and time required in generating models with desired genetic modifications. In this regard, the ability to directly modify the genome in the zygote and generate edited animals is highly desirable. This report demonstrates for the first time, the generation of gene targeted animals by direct injection of Cas9 ribonucleoprotein complex and short stretches of DNA sequences into porcine zygotes. The Cas9 protein from Streptococcus pyogenes was pre-complexed with a single guide RNA targeting downstream of the ubiquitously expressed COL1A gene, and co-injected with a single-stranded repair template into porcine zygotes. Using this approach a line of pigs that carry pseudo attP sites within the COL1A locus to enable phiC31 integrase mediated introduction of transgenes has been generated. This new route for genome engineering in pigs via zygote injection should greatly enhance applications in both agriculture and biomedicine.

The creation of genetically modified goats provides a powerful approach for improving animal health, enhancing production traits, animal pharming, and for ensuring food safety all of which are high-priority goals for animal agriculture. The availability of goat embryonic stem cells (ESCs) that are characteristically immortal in culture would be of enormous benefit for developing genetically modified animals. As an alternative to long-sought goat ESCs, we generated induced pluripotent stem cells (iPSC) by forced expression of bovine POU5F1, SOX2, MYC, KLF4, LIN-28, and NANOG reprogramming factors in combination with a MIR302/367 cluster, delivered by lentiviral vectors. In order to minimize integrations, the reprogramming factor coding sequences were assembled with porcine teschovirus-1 2A (P2A) self-cleaving peptides that allowed for tri-cistronic expression from each vector. The lentiviral-transduced cells were cultured on irradiated mouse feeder cells in a semi-defined, serum-free medium containing fibroblast growth factor (FGF) and/or leukemia inhibitory factor (LIF). The resulting goat iPSC exhibit cell and colony morphology typical of human and mouse ESCs-that is, well-defined borders, a high nuclear-to-cytoplasmic ratio, a short cell-cycle interval, alkaline phosphatase expression, and the ability to generate teratomas in vivo. Additionally, these goat iPSC demonstrated the ability to differentiate into directed lineages in vitro. These results constitute the first steps in establishing integration and footprint-free iPSC from ruminants. Mol. Reprod. Dev. 82: 709-721, 2015. © 2015 Wiley Periodicals, Inc.

MicroRNAs or miRNAs belong to a class of small noncoding RNAs that play a crucial role in posttranscriptional regulation of gene expression. Nascent miRNAs are expressed as a longer transcript, which are then processed into a smaller 18-23-nucleotide mature miRNAs that bind to the target transcripts and induce cleavage or inhibit translation. MiRNAs therefore represent another key regulator of gene expression in establishing and maintaining unique cellular fate. Several classes of miRNAs have been identified to be uniquely expressed in embryonic stem cells (ESC) and regulated by the core transcription factors Oct4, Sox2, and Klf4. One such class of miRNAs is the mir-302/367 cluster that is enriched in pluripotent cells in vivo and in vitro. Using the mir-302/367 either by themselves or in combination with the Yamanaka reprogramming factors (Oct4, Sox2, c-Myc, and Klf4) has resulted in the establishment of induced pluripotent stem cells (iPSC) with high efficiencies. In this chapter, we outline the methodologies for establishing and utilizing the miRNA-based tools for reprogramming somatic cells into iPSC.

Philippine crocodiles Crocodylus mindorensis are critically endangered due to agricultural and fishing threats that have severely fragmented their habitat and population in the Philippines. Captive management plans are important to safeguard against their extinction, but the current population in US zoos is small, and breeding is hampered by the slow growth of this species and the danger of introducing differently sized animals for breeding. There is little information regarding the sperm characteristics of crocodilians, and none for Philippine crocodiles. In this study, we sought to characterise sperm production in the male Philippine crocodile (n = 1) by performing voluntary (without sedation or restraint) collections (n = 181) over a 3.5-year period. Peak sperm production in this individual occurs from January to July, when the mean (±s.e.m.) total number of spermatozoa recovered was 10.2 × 106 ± 3.8 × 106 (n = 104), compared with 0.3 × 106 ± 0.2 × 106 (n = 71) for all other months of the year. Analysis of sperm morphology indicated that 15.9% of spermatozoa exhibited normal morphology. A bent tail was the most common abnormality (48.2%) observed. Understanding the basic reproductive biology of the male Philippine crocodile will facilitate the development of artificial reproductive technologies to improve captive propagation and genetic management of this species.