No abstract

Although pigs are used widely as models of human disease, their utility as models has been enhanced by genetic engineering. Initially, transgenes were added randomly to the genome, but with the application of homologous recombination, zinc finger nucleases, and transcription activator-like effector nuclease (TALEN) technologies, now most any genetic change that can be envisioned can be completed. To date these genetic modifications have resulted in animals that have the potential to provide new insights into human diseases for which a good animal model did not exist previously. These new animal models should provide the preclinical data for treatments that are developed for diseases such as Alzheimer's disease, cystic fibrosis, retinitis pigmentosa, spinal muscular atrophy, diabetes, and organ failure. These new models will help to uncover aspects and treatments of these diseases that were otherwise unattainable. The focus of this review is to describe genetically engineered pigs that have resulted in models of human diseases.

Successful divisions of eukaryotic cells require accurate and coordinated cycles of DNA replication, spindle formation, chromosome segregation, and cytoplasmic cleavage. The Caenorhabditis elegans gene lin-5 is essential for multiple aspects of cell division. Cells in lin-5 null mutants enter mitosis at the normal time and form bipolar spindles, but fail chromosome alignment at the metaphase plate, sister chromatid separation, and cytokinesis. Despite these defects, cells exit from mitosis without delay and progress through subsequent rounds of DNA replication, centrosome duplication, and abortive mitoses. In addition, early embryos that lack lin-5 function show defects in spindle positioning and cleavage plane specification. The lin-5 gene encodes a novel protein with a central coiled-coil domain. This protein localizes to the spindle apparatus in a cell cycle- and microtubule-dependent manner. The LIN-5 protein is located at the centrosomes throughout mitosis, at the kinetochore microtubules in metaphase cells, and at the spindle during meiosis. Our results show that LIN-5 is a novel component of the spindle apparatus required for chromosome and spindle movements, cytoplasmic cleavage, and correct alternation of the S and M phases of the cell cycle.

Alternative splicing emerges as one of the most important mechanisms to generate transcript diversity. It is regulated by the formation of protein complexes on pre-mRNA. We demonstrate that protein phosphatase 1 (PP1) binds to the splicing factor transformer2-beta1 (tra2-beta1) via a phylogenetically conserved RVDF sequence located on the RNA recognition motif (RRM) of tra2-beta1. PP1 binds directly to tra2-beta1 and dephosphorylates it, which regulates the interaction between tra2-beta1 and other proteins. Eight other proteins, including SF2/ASF and SRp30c, contain an evolutionary conserved PP1 docking motif in the beta-4 strand of their RRMs indicating that binding to PP1 is a new function of some RRMs. Reducing PP1 activity promotes usage of numerous alternative exons, demonstrating a role of PP1 activity in splice site selection. PP1 inhibition promotes inclusion of the survival of motoneuron 2 exon 7 in a mouse model expressing the human gene. This suggests that reducing PP1 activity could be a new therapeutic principle to treat spinal muscular atrophy and other diseases caused by missplicing events. Our data indicate that the binding of PP1 to evolutionary conserved motifs in several RRMs is the link between known signal transduction pathways regulating PP1 activity and pre-mRNA processing.

Mitotic cyclins in association with the Cdk1 protein kinase regulate progression through mitosis in all eukaryotes. Here, we address to what extent mitotic cyclins in the nematode Caenorhabditis elegans provide overlapping functions or distinct biological activities. C. elegans expresses a single A-type cyclin (CYA-1), three typical B-type cyclins (CYB-1, CYB-2.1 and CYB-2.2), and one B3-subfamily member (CYB-3). While we observed clear redundancies between the cyb genes, cyb-1 and cyb-3 also contribute specific essential functions in meiosis and mitosis. CYB-1 and CYB-3 show similar temporal and spatial expression, both cyclins localize prominently to the nucleus, and both associate with CDK-1 and display histone H1 kinase activity in vitro. We demonstrate that inhibition of cyb-1 by RNAi interferes with chromosome congression and causes aneuploidy. In contrast, cyb-3(RNAi) embryos fail to initiate sister chromatid separation. Inhibition of both cyclins simultaneously results in a much earlier and more dramatic arrest. However, only the combination of cyb-1, cyb-3 and cyb-2.1/cyb-2.2 RNAi fully resembles cdk-1 inhibition. This combination of redundant and specific phenotypes supports that in vivo phosphorylation of certain Cdk targets can be achieved by multiple Cdk1/cyclin complexes, while phosphorylation of other targets requires a unique Cdk1/cyclin combination.