An analysis of aspartic peptidases expressed by trophoblasts and placenta of even-toed ungulates

Title from PDF of title page (University of Missouri--Columbia, viewed on February 23, 2010).The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file.Dissertation advisor: Dr. Jonathan A. Green.VitaPh.D. University of Missouri--Columbia 2008.The Pregnancy Associated Glycoproteins (PAGs) represent a multigene family of trophoblast expressed proteins, found exclusively in the placenta of even-toed mammals such as ruminants (cattle, sheep), pig, etc. In ruminants, the PAGs can be classified into ancient and modern PAGs based on their coding sequence. In addition, there are also differences in purported enzymatic activity as well as transcriptional regulation of expression. Many of the modern PAGs have accumulated mutations in and around the catalytic center, and some of those that incurred mutations in the two catalytic aspartates are predicted to be proteolytically inactive. In contrast, most of the ancient PAGs of ruminants and swine, have all the hallmarks of typical aspartic peptidases (APs). From the analysis of cattle genome, we found that there are 18 distinct PAG genes and 14 pseudogenes. Based on our preliminary analysis of the proximal promoter regions [500 base pairs (bp) upstream of the translational start point] of PAG genes, we found that there are pockets of conserved transcription factor binding sites that are different between ancient and modern PAGs. These differences likely influence the observed differences in expression between ancient and modern boPAGs. We gathered evidence by Real-time PCR and global analysis of expressed ESTs that confirm that, boPAG-2 is the most abundant of all boPAGs. We identified boPAG-2 and its closest paralog boPAG-12, as well as poPAG-2 the ancient PAG found in pigs, as the candidates for investigation of proteolytic activity. From our experiments we found that, boPAGs -2 and -12 and poPAG-2 are proteases with optimal activity under acidic pH conditions. We also illustrated differences in proteolytic activity towards substrates, and in their relative affinity towards an AP inhibitor (pepstatin A). We found that, in comparison to the two bovine paralogs, boPAGs -2 and -12, poPAG-2 was found to be a more robust enzyme. Finally, we demonstrated that APs secreted by embryos such as PAGs can be objectively measured in the medium conditioned by the culture of porcine embryos either individually or in pools for variable lengths of time in acidic conditions. We also observed that such activity seemed to correlate with stage and quality of embryos (assessed morphologically) in vitro. We, therefore, believe that this proteolytic activity potentially could serve as a marker for developmental competence of the embryos.Includes bibliographical references

Characterization of the bovine pregnancy-associated glycoprotein gene family – analysis of gene sequences, regulatory regions within the promoter and expression of selected genes

<p>Abstract</p> <p>Background</p> <p>The Pregnancy-associated glycoproteins (PAGs) belong to a large family of aspartic peptidases expressed exclusively in the placenta of species in the <it>Artiodactyla </it>order. In cattle, the <it>PAG </it>gene family is comprised of at least 22 transcribed genes, as well as some variants. Phylogenetic analyses have shown that the PAG family segregates into 'ancient' and 'modern' groupings. Along with sequence differences between family members, there are clear distinctions in their spatio-temporal distribution and in their relative level of expression. In this report, 1) we performed an <it>in silico </it>analysis of the bovine genome to further characterize the <it>PAG </it>gene family, 2) we scrutinized proximal promoter sequences of the <it>PAG </it>genes to evaluate the evolution pressures operating on them and to identify putative regulatory regions, 3) we determined relative transcript abundance of selected <it>PAGs </it>during pregnancy and, 4) we performed preliminary characterization of the putative regulatory elements for one of the candidate PAGs, <it>bovine </it>(<it>bo</it>) <it>PAG-2</it>.</p> <p>Results</p> <p>From our analysis of the bovine genome, we identified 18 distinct <it>PAG </it>genes and 14 pseudogenes. We observed that the first 500 base pairs upstream of the translational start site contained multiple regions that are conserved among all <it>boPAGs</it>. However, a preponderance of conserved regions, that harbor recognition sites for putative transcriptional factors (TFs), were found to be unique to the modern <it>boPAG </it>grouping, but not the ancient <it>boPAGs</it>. We gathered evidence by means of Q-PCR and screening of EST databases to show that <it>boPAG-2 </it>is the most abundant of all <it>boPAG </it>transcripts. Finally, we provided preliminary evidence for the role of ETS- and DDVL-related TFs in the regulation of the <it>boPAG-2 </it>gene.</p> <p>Conclusion</p> <p><it>PAGs </it>represent a relatively large gene family in the bovine genome. The proximal promoter regions of these genes display differences in putative TF binding sites, likely contributing to observed differences in spatial and temporal expression. We also discovered that <it>boPAG-2 </it>is the most abundant of all boPAG transcripts and provided evidence for the role of ETS and DDVL TFs in its regulation. These experiments mark the crucial first step in discerning the complex transcriptional regulation operating within the <it>boPAG </it>gene family.</p

Derivation and characterization of LIF and FGF2 dependent Porcine Induced Pluripotent Stem Cells [abstract]

Comparative Medicine - OneHealth and Comparative Medicine Poster SessionAuthentic embryonic stem cells (ESC) have probably never been derived from the inner cell mass (ICM) of pig, despite over 25 years of effort. Recently, several groups, including ours, have reported derivation of induced pluripotent stem cells (iPSC) from swine by reprogramming somatic cells with a combination of four factors (OCT4, SOX2, KLF4, C-MYC) delivered by retroviral transduction. The piPSC resembled FGF2- dependent human (h) ESC and are likely to advance swine as a model in biomedical research, since grafts could potentially be matched to the animal that donated the cells for re-programming. The dependence of piPSC on FGF2 also draws parallels to murine pluripotent stem cells derived from advanced epiblast, so-called 'epiblast stem cells'. Indeed, an emerging concept is that there are two kinds of ESC, one dependent on FGF2 and not competent to contribute to germ-line chimeras, the other upon LIF and germ-line competent. The objective of our recent investigations has been to develop LIF- dependent piPSC by using the same reprogramming factors but selecting the colonies on a modified LIF- medium supplemented with two kinase inhibitors, CHIR99021, which inhibits GSK-3beta, and kenpaullone, which inhibits both GSK-3beta and CDK1. The LIF-dependent piPSC, derived here from outgrowths of umbilical cord mesenchyme, expressed markers consistent with pluripotency and bore a striking resemblance to ICM-derived murine ESC in colony morphology, culture characteristics, and short cell cycle time. Currently, the ability of LIF-piPSC to give rise to teratoma and chimeras is under investigation. Supported by Missouri Life Sciences Board Grant 00022147 and NIH grant HD21896

Porcine induced pluripotent stem cells (piPSC) for expanding the use of swine in biomedical research

Comparative Medicine - OneHealth and Comparative Medicine Poster SessionOur goal is to create porcine pluripotent stem cells, i.e. ones capable of differentiating into all cell types of the body that can expand the use of swine as a biomedical model for studying human disease. It is well established that mouse embryonic stem cells (ESC) are an excellent source of material for successful cloning and for incorporation into chimeras. However, the establishment of porcine ESC from the embryos has proven to be elusive. There has been a similar lack of success with other ungulate species. Establishing a technology for deriving induced pluripotent stem cells (iPSC) from farm animals will allow the gene knock-in/knock-out methods that have revolutionized mouse genetics to be applied to farm species. Importantly pig is a potentially useful model for studying human pathologies due to similarities in organ size, immunology and whole animal physiology between the two species. If the safety and efficacy of stem cell transplantation is to be tested in an animal model before being applied to humans, the pig would likely be a species of choice. The ability to derive porcine (p) iPSC lines from a particular outbred animal and conduct tissue transplantation on the same pig later and follow the success of the transplant over the course of months or even years would be a particularly valuable advance. Additionally the ability to provide piPSC from animals with valuable traits would provide a permanent source of cells for clonal propagation that would likely avoid the inefficiencies and problems arising from somatic cell nuclear transfer (SCNT), where the vast majority of cloned offspring die or are developmentally abnormal. We have created piPSC from embryonic fibroblasts and umbilical cord mesenchyme by a similar strategy used for the mouse and human, namely ectopically expressing reprogramming genes in somatic cells. The piPSC resemble human ESC, express the typical gene and surface antigen markers of ESC, proliferate continuously in culture, possess high telomerase activity, form embryoid bodies, and differentiate along the three main germ line lineages. Our aim is to demonstrate that piPSC can be directed to differentiate along defined lineages, specifically towards neuronal tissue, hematopoietic lineages and various mesoderm derivatives including cardiomyocytes by using protocols based on those used successfully with human and murine ESC. These experiments will allow such cells to be used for tissue grafts that are matched genetically to recipients and tested for their safety in transplantation. We shall also establish parameters for routine gene targeting in piPSC, with the ultimate goal of creating genetic models for human diseases where mouse models are inappropriate. In summary, the piPSC lines developed will have enormous utility for exploiting the pig as a model in human pre-clinical applications. Supported by Missouri Life Sciences Board Grant 00022147 and NIH grant HD2189

Engineering large animal models of human disease:Domesticated Animal Models of Human Disease

The recent development of gene editing tools and methodology for use in livestock enables the production of new animal disease models. These tools facilitate site‐specific mutation of the genome, allowing animals carrying known human disease mutations to be produced. In this review, we describe the various gene editing tools and how they can be used for a range of large animal models of diseases. This genomic technology is in its infancy but the expectation is that through the use of gene editing tools we will see a dramatic increase in animal model resources available for both the study of human disease and the translation of this knowledge into the clinic. Comparative pathology will be central to the productive use of these animal models and the successful translation of new therapeutic strategies. © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland

Genome to Phenome: Improving Animal Health, Production, and Well-Being – A New USDA Blueprint for Animal Genome Research 2018–2027

In 2008, a consortium led by the Agricultural Research Service (ARS) and the National Institute for Food and Agriculture (NIFA) published the “Blueprint for USDA Efforts in Agricultural Animal Genomics 2008–2017,” which served as a guiding document for research and funding in animal genomics. In the decade that followed, many of the goals set forth in the blueprint were accomplished. However, several other goals require further research. In addition, new topics not covered in the original blueprint, which are the result of emerging technologies, require exploration. To develop a new, updated blueprint, ARS and NIFA, along with scientists in the animal genomics field, convened a workshop titled “Genome to Phenome: A USDA Blueprint for Improving Animal Production” in November 2017, and these discussions were used to develop new goals for the next decade. Like the previous blueprint, these goals are grouped into the broad categories “Science to Practice,” “Discovery Science,” and “Infrastructure.” New goals for characterizing the microbiome, enhancing the use of gene editing and other biotechnologies, and preserving genetic diversity are included in the new blueprint, along with updated goals within many genome research topics described in the previous blueprint. The updated blueprint that follows describes the vision, current state of the art, the research needed to advance the field, expected deliverables, and partnerships needed for each animal genomics research topic. Accomplishment of the goals described in the blueprint will significantly increase the ability to meet the demands for animal products by an increasing world population within the next decade

Genome editing to the rescue: sustainably feeding 10 billion global human population

Modern animal breeding strategies based on population genetics, molecular tools, artificial insemination, embryo transfer and related technologies have contributed to significant increases in the performance of domestic animals, and are the basis for a regular supply of high quality animal derived food at acceptable prices. However, the current strategy of marker- assisted selection and breeding of animals to introduce novel traits over multiple generations is too pedestrian in responding to unprecedented challenges such as climate change, global pandemics, and feeding an anticipated 33% increase in global population in the next three decades. Here, we propose site-specific genome editing technologies as a basis for “directed” or “rational selection” of agricultural traits. The animal science community envisions genome editing as an essential tool in addressing critical priorities for global food security and environmental sustainability, and seeks additional funding support for development and implementation of these technologies for maximum societal benefit

Genome to Phenome: Improving Animal Health, Production, and Well-Being – A New USDA Blueprint for Animal Genome Research 2018–2027

In 2008, a consortium led by the Agricultural Research Service (ARS) and the National Institute for Food and Agriculture (NIFA) published the “Blueprint for USDA Efforts in Agricultural Animal Genomics 2008–2017,” which served as a guiding document for research and funding in animal genomics. In the decade that followed, many of the goals set forth in the blueprint were accomplished. However, several other goals require further research. In addition, new topics not covered in the original blueprint, which are the result of emerging technologies, require exploration. To develop a new, updated blueprint, ARS and NIFA, along with scientists in the animal genomics field, convened a workshop titled “Genome to Phenome: A USDA Blueprint for Improving Animal Production” in November 2017, and these discussions were used to develop new goals for the next decade. Like the previous blueprint, these goals are grouped into the broad categories “Science to Practice,” “Discovery Science,” and “Infrastructure.” New goals for characterizing the microbiome, enhancing the use of gene editing and other biotechnologies, and preserving genetic diversity are included in the new blueprint, along with updated goals within many genome research topics described in the previous blueprint. The updated blueprint that follows describes the vision, current state of the art, the research needed to advance the field, expected deliverables, and partnerships needed for each animal genomics research topic. Accomplishment of the goals described in the blueprint will significantly increase the ability to meet the demands for animal products by an increasing world population within the next decade