Creating genetically modified pigs by using nuclear transfer

Nuclear transfer (NT) is a procedure by which genetically identical individuals can be created. The technology of pig somatic NT, including in vitro maturation of oocytes, isolation and treatment of donor cells, artificial activation of reconstructed oocytes, embryo culture and embryo transfer, has been intensively studied in recent years, resulting in birth of cloned pigs in many labs. While it provides an efficient method for producing transgenic pigs, more importantly, it is the only way to produce gene-targeted pigs. So far pig cloning has been successfully used to produce transgenic pigs expressing the green fluorescence protein, expand transgenic pig groups and create gene targeted pigs which are deficient of alpha-1,3-galactosyltransferase. The production of pigs with genetic modification by NT is now in the transition from investigation to practical use. Although the efficiency of somatic cell NT in pig, when measured as development to term as a proportion of oocytes used, is not high, it is anticipated that the ability of making specific modifications to the swine genome will result in this technology having a large impact not only on medicine but also on agriculture

Generation of cloned transgenic pigs rich in omega-3 fatty acids

Meat products are generally low in omega-3 (n-3) fatty acids, which are beneficial to human health. We describe the generation of cloned pigs that express a humanized Caenorhabditis elegans gene, fat-1, encoding an n-3 fatty acid desaturase. The hfat-1 transgenic pigs produce high levels of n-3 fatty acids from n-6 analogs, and their tissues have a significantly reduced ratio of n-6/n-3 fatty acids (P < 0.001). © 2006 Nature Publishing Group

Use of the 2A Peptide for Generation of Multi-Transgenic Pigs through a Single Round of Nuclear Transfer

Multiple genetic modifications in pigs can essentially benefit research on agriculture, human disease and xenotransplantation. Most multi-transgenic pigs have been produced by complex and time-consuming breeding programs using multiple single-transgenic pigs. This study explored the feasibility of producing multi-transgenic pigs using the viral 2A peptide in the light of previous research indicating that it can be utilized for multi-gene transfer in gene therapy and somatic cell reprogramming. A 2A peptide-based double-promoter expression vector that mediated the expression of four fluorescent proteins was constructed and transfected into primary porcine fetal fibroblasts. Cell colonies (54.3%) formed under G418 selection co-expressed the four fluorescent proteins at uniformly high levels. The reconstructed embryos, which were obtained by somatic cell nuclear transfer and confirmed to express the four fluorescent proteins evenly, were transplanted into seven recipient gilts. Eleven piglets were delivered by two gilts, and seven of them co-expressed the four fluorescent proteins at equivalently high levels in various tissues. The fluorescence intensities were directly observed at the nose, hoof and tongue using goggles. The results suggest that the strategy of combining the 2A peptide and double promoters efficiently mediates the co-expression of the four fluorescent proteins in pigs and is hence a promising methodology to generate multi-transgenic pigs by a single nuclear transfer

Fumarylacetoacetate Hydrolase Knock-out Rabbit Model for Hereditary Tyrosinemia Type 1.

Hereditary tyrosinemia type 1 (HT1) is a severe human autosomal recessive disorder caused by the deficiency of fumarylacetoacetate hydroxylase (FAH), an enzyme catalyzing the last step in the tyrosine degradation pathway. Lack of FAH causes accumulation of toxic metabolites (fumarylacetoacetate and succinylacetone) in blood and tissues, ultimately resulting in severe liver and kidney damage with onset that ranges from infancy to adolescence. This tissue damage is lethal but can be controlled by administration of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), which inhibits tyrosine catabolism upstream of the generation of fumarylacetoacetate and succinylacetone. Notably, in animals lacking FAH, transient withdrawal of NTBC can be used to induce liver damage and a concomitant regenerative response that stimulates the growth of healthy hepatocytes. Among other things, this model has raised tremendous interest for the in vivo expansion of human primary hepatocytes inside these animals and for exploring experimental gene therapy and cell-based therapies. Here, we report the generation of FAH knock-out rabbits via pronuclear stage embryo microinjection of transcription activator-like effector nucleases. FAH-/- rabbits exhibit phenotypic features of HT1 including liver and kidney abnormalities but additionally develop frequent ocular manifestations likely caused by local accumulation of tyrosine upon NTBC administration. We also show that allogeneic transplantation of wild-type rabbit primary hepatocytes into FAH-/- rabbits enables highly efficient liver repopulation and prevents liver insufficiency and death. Because of significant advantages over rodents and their ease of breeding, maintenance, and manipulation compared with larger animals including pigs, FAH-/- rabbits are an attractive alternative for modeling the consequences of HT1.Wellcome Trus

Felodipine induces autophagy in mouse brains with pharmacokinetics amenable to repurposing.

Neurodegenerative diseases like Alzheimer's disease, Parkinson's disease and Huntington's disease manifest with the neuronal accumulation of toxic proteins. Since autophagy upregulation enhances the clearance of such proteins and ameliorates their toxicities in animal models, we and others have sought to re-position/re-profile existing compounds used in humans to identify those that may induce autophagy in the brain. A key challenge with this approach is to assess if any hits identified can induce neuronal autophagy at concentrations that would be seen in humans taking the drug for its conventional indication. Here we report that felodipine, an L-type calcium channel blocker and anti-hypertensive drug, induces autophagy and clears diverse aggregate-prone, neurodegenerative disease-associated proteins. Felodipine can clear mutant α-synuclein in mouse brains at plasma concentrations similar to those that would be seen in humans taking the drug. This is associated with neuroprotection in mice, suggesting the promise of this compound for use in neurodegeneration

Stem Cells for Huntington's Disease (SC4HD): An International Consortium to Facilitate Stem Cell-Based Therapy for Huntington's Disease

Huntington's disease (HD) research is entering an exciting phase, with new approaches such as huntingtin lowering strategies and cell therapies on the horizon. Technological advances to direct the differentiation of stem cells to desired neural types have opened new strategies for restoring damaged neuronal circuits in HD. However, challenges remain in the implementation of cell therapy approaches for patients suffering from HD. Cell therapies, together with other invasive approaches including allele specific oligonucleotides (ASOs) and viral delivery of huntingtin-lowering agents, require direct delivery of the therapeutic agents locally into the brain or cerebrospinal fluid. Delivering substances directly into the brain is complex and presents multiple challenges, including those related to regulatory requirements, safety and efficacy, surgical instrumentation, trial design, patient profiles, and selection of suitable and sensitive primary and secondary outcomes. In addition, production of clinical grade cell-based medicinal products also requires adherence to regulatory standards with extensive quality control of the protocols and cell products across different laboratories and production centers. Currently, there is no consensus on how best to address these challenges. Here we describe the formation of Stem Cells For Huntington's Disease (SC4HD: https://www.sc4hd.org/), a network of researchers and clinicians working to develop guidance and greater standardization for the HD field for stem cell based transplantation therapy for HD with a mission to work to develop criteria and guidance for development of a neural intra-cerebral stem cell-based therapy for HD

Loss of Angiopoietin-like 7 diminishes the regeneration capacity of hematopoietic stem and progenitor cells

© 2015 Xiao et al.; licensee Biomed Central. Successful expansion of hematopoietic stem cells (HSCs) would benefit the use of HSC transplants in the clinic. Angiopoietin-like 7 promotes the expansion of hematopoietic stem and progenitor cells (HSPC) in vitro and ex vivo. However, the impact of loss of Angptl7 on HSPCs in vivo has not been characterized. Here, we generated Angptl7-deficient mice by TALEN-mediated gene targeting and found that HSC compartments in Angptl7-null mice were compromised. In addition, wild type (WT) HSPCs failed to repopulate in the BM of Angptl7-null mice after serial transplantations while the engraftment of Angptl7-deficient HSPCs in WT mice was not impaired. These results suggest that Angptl7 is required for HSPCs repopulation in a non-cell autonomous manner.Link_to_subscribed_fulltex

Stem cells for Huntington’s disease (SC4HD): an international consortium to facilitate stem cell-based therapy for Huntington’s disease

Huntington’s disease (HD) research is entering an exciting phase, with new approaches such as huntingtin lowering strategies and cell therapies on the horizon. Technological advances to direct the differentiation of stem cells to desired neural types have opened new strategies for restoring damaged neuronal circuits in HD. However, challenges remain in the implementation of cell therapy approaches for patients suffering from HD. Cell therapies, together with other invasive approaches including allele specific oligonucleotides (ASOs) and viral delivery of huntingtin-lowering agents, require direct delivery of the therapeutic agents locally into the brain or cerebrospinal fluid. Delivering substances directly into the brain is complex and presents multiple challenges, including those related to regulatory requirements, safety and efficacy, surgical instrumentation, trial design, patient profiles, and selection of suitable and sensitive primary and secondary outcomes. In addition, production of clinical grade cell-based medicinal products also requires adherence to regulatory standards with extensive quality control of the protocols and cell products across different laboratories and production centers. Currently, there is no consensus on how best to address these challenges. Here we describe the formation of Stem Cells For Huntington’s Disease (SC4HD: https://www.sc4hd.org/), a network of researchers and clinicians working to develop guidance and greater standardization for the HD field for stem cell based transplantation therapy for HD with a mission to work to develop criteria and guidance for development of a neural intra-cerebral stem cell-based therapy for HD

Establishment of porcine and human expanded potential stem cells.

We recently derived mouse expanded potential stem cells (EPSCs) from individual blastomeres by inhibiting the critical molecular pathways that predispose their differentiation. EPSCs had enriched molecular signatures of blastomeres and possessed developmental potency for all embryonic and extra-embryonic cell lineages. Here, we report the derivation of porcine EPSCs, which express key pluripotency genes, are genetically stable, permit genome editing, differentiate to derivatives of the three germ layers in chimeras and produce primordial germ cell-like cells in vitro. Under similar conditions, human embryonic stem cells and induced pluripotent stem cells can be converted, or somatic cells directly reprogrammed, to EPSCs that display the molecular and functional attributes reminiscent of porcine EPSCs. Importantly, trophoblast stem-cell-like cells can be generated from both human and porcine EPSCs. Our pathway-inhibition paradigm thus opens an avenue for generating mammalian pluripotent stem cells, and EPSCs present a unique cellular platform for translational research in biotechnology and regenerative medicine