Induced Pluripotent Stem Cells from Swine: application to genetic modification

猪诱导多能干细胞：在基因改造中的应用

• 批准号: 8183121
• 负责人: R. MICHAEL ROBERTS
• 金额: $ 30.77万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-01 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8183121
• 关键词: Abbreviations; Agriculture; Alleles; Atherosclerosis; Azacitidine; Biomedical Research; Capital; Cardiovascular Diseases; Cell Culture Techniques; Cell Cycle; Cell Line; Cell Nucleus; Cells; Chimera organism; Cloning; Communities; Coronary heart disease; Cytoplasm; Development; Diabetes Mellitus; Diet; Domestic Fowls; ES Cell Line; Embryo; Embryo Transfer; Employee Strikes; Epigenetic Process; Excision; Exhibits; FGF2 gene; Family suidae; Fetus; Fiber; Fibroblast Growth Factor; Fibroblasts; Fluorescence; Gene Combinations; Gene Targeting; Gene Transfer Techniques; Genes; Genetic; Genetically Modified Animals; Germ Lines; Goals; Growth; Heterozygote; Homozygote; Human; In Vitro; Industry; Inner Cell Mass; Intestines; Knock-in Mouse; Knock-out; LIF gene; Laboratories; Lentivirus Vector; Lipoproteins; Livestock; Low Density Lipoprotein Receptor; Low-Density Lipoproteins; Malignant Neoplasms; Meat; Medicine; Memory; Mesenchyme; Metabolic syndrome; Metabolism; Metallothionein I; Microinjections; Milk; Missouri; Modeling; Modification; Morphology; Mus; Mutation; Nuclear; Onset of illness; Oocytes; Ophthalmology; Organ; Outcome; Pharmaceutical Preparations; Pharmacologic Substance; Pregnancy loss; Procedures; Process; Production; Proliferating; Proteins; Protocols documentation; Receptor Gene; Research; Resources; Salmonella; Salmonella infections; Science; Serum; Somatic Cell; Somatropin; Source; Staging; Technology; Teratoma; Testing; Tetanus Helper Peptide; Toxicology; Transfection; Transgenes; Transgenic Organisms; Trypsin; Umbilical cord structure; Undifferentiated; United States National Institutes of Health; Universities; Ursidae Family; Wound Healing; Xenograft procedure; blastocyst; cell type; embryonic stem cell; fetal; genetic resource; homologous recombination; hypercholesterolemia; improved; in vivo; induced pluripotent stem cell; injury and repair; interest; juvenile animal; loss of function; neonate; nuclear transfer; offspring; pluripotency; promoter; pup; research study; response; retroviral transduction; somatic cell nuclear transfer; success; vector; zygote

DESCRIPTION (provided by applicant): The goal here is to utilize induced pluripotent stem cells (iPSC) derived from the inner cell mass (ICM) of porcine blastocysts to clone genetically modified pigs by nuclear transfer (NT) procedures. In our laboratory, the ICM-derived iPSC so far generated are LIF-dependent, have short cell cycle intervals and resemble mouse embryonic stem cells (ESC). They can survive multiple passages without senescing and are, therefore, suitable for introduction of multiple genetic changes under extended pharmacological selection. As the cells are pluripotent and "undifferentiated", and possibly carry the epigenetic "memory" of the ICM from which they originated, they may be more easily reprogrammed during cloning than other somatic cell types and provide fewer abnormalities in offspring born. Accordingly, these cells are likely to have considerable potential for manipulating porcine genetics and hence be of value to the livestock industry and to the biomedical research in general. Aim 1 is to establish additional lines of LIF-dependent ICM-derived porcine (p) iPSC (pICM-iPSC) by upregulating human POU5F1 and KLF4 transgenes assembled on "tet-on" bicistronic lentiviral vectors, which offer the advantage of effective transgene silencing or removal, and characterize the resulting lines in further detail. Lines that grow vigorously and demonstrate full in vitro and in vivo (teratoma) criteria for pluripotency will then be tested for their ability to participate in chimera production. Such chimeras may also be valuable as an alternative to cloning for creating genetically modified swine. Aim 2 is to determine whether or not the various LIF-and FGF2-dependent piPSC at our disposal are superior donors than somatic cells for nuclear transfer. This aim will also allow us to test if there is any difference between the piPSC representing different classes of pluripotency (LIF-and FGF-dependent) and between the pICM-iPSC and analogous LIF-dependent piPSC reprogrammed from somatic cells. A sub-aim will be to verify if the offspring born to pICM-iPSC show fewer developmental abnormalities compared to the somatic cells by nuclear transfer. Aim 3 is to establish parameters for efficient gene targeting and transgenesis in pICM-iPSC. Transfection procedures and the effective concentration ranges of selection agents and targeting vectors used to introduce a knockout of one of the alleles of the gene encoding the receptor for low density lipoproteins (LDLR) by homologous recombination will be evaluated. Aim 4 is to delete both alleles of LDLR and thereby produce a genetically modified pig with potential interest to both agriculture and medicine by NT. Loss of function of LDLR in humans causes hypercholesterolemia, coronary heart disease, and onset of the so-called metabolic syndrome. Pigs lacking one or both copies will provide an alternative model to the mouse for studying responses to diet and drugs in treatment of these conditions. LDLR deficiency and associated hypercholesterolemia are also postulated to protect against Salmonella infections. As the pig is an important reservoir for Salmonella, the LDLR-/- model will have a dual purpose for both agricultural and biomedical applications. PUBLIC HEALTH RELEVANCE: The purpose of this proposal is to utilize lines of induced pluripotent stem cells (iPSC) derived from the inner cell mass (ICM) of porcine blastocysts to produce genetically modified pigs by nuclear transfer, i.e. cloning, procedures. Accordingly, these cells are likely to have considerable potential for manipulating porcine genetics and hence be of value to both the livestock industry and to the biomedical sciences.

描述(申请人提供)：这里的目标是利用从猪囊胚内细胞团(ICM)中提取的诱导多能干细胞(IPSC)通过核移植(NT)程序克隆转基因猪。在我们的实验室中，到目前为止所产生的ICM来源的IPSC是LIF依赖的，具有短的细胞周期间隔，并且类似于小鼠胚胎干细胞(ESC)。它们可以在多代传代中存活而不衰老，因此，适合于在扩展的药理选择下引入多种基因变化。由于这些细胞是多能的和未分化的，而且可能携带它们起源的ICM的表观遗传“记忆”，它们在克隆过程中可能比其他类型的体细胞更容易重新编程，在出生的后代中出现的异常较少。因此，这些细胞很可能有相当大的潜力来操纵猪的遗传学，因此对畜牧业和一般的生物医学研究都有价值。目的1是通过上调组装在“tet-on”双顺反子慢病毒载体上的人Pou5f1和KLF4转基因，建立依赖LIF的ICM来源的猪(P)IPSC(PICM-IPSC)的附加系，并进一步详细描述所得到的系。然后，将测试生长旺盛并在体外和体内(畸胎瘤)完全符合多能性标准的品系，以确定它们参与嵌合体生产的能力。这种嵌合体作为克隆创造转基因猪的替代方案也可能是有价值的。目的2是确定我们所使用的各种依赖LIF和FGF2的PIPSC是否是比体细胞更好的核移植供体。这一目标也将使我们能够测试代表不同类别的多能性(依赖LIF和成纤维细胞生长因子)的PIPSC之间，以及PICM-IPSC和从体细胞重新编程的类似的依赖LIF的PIPSC之间是否存在任何差异。下一个目标将是验证通过核移植出生的PICM-IPSC后代是否表现出比体细胞更少的发育异常。目的3是建立有效的PICM-IPSC基因打靶和转基因参数。将评估用于通过同源重组引入低密度脂蛋白受体(LDLR)编码基因的一个等位基因敲除的转染法和选择剂和靶向载体的有效浓度范围。目的4是删除LDLR的两个等位基因，从而通过NT培育出一种兼具农业和医药双重价值的转基因猪。人类低密度脂蛋白受体功能的丧失会导致高胆固醇血症、冠心病和所谓的代谢综合征的发生。缺乏一个或两个副本的猪将为研究治疗这些疾病的饮食和药物反应的小鼠提供另一种模型。低密度脂蛋白受体缺乏和相关的高胆固醇血症也被认为可以预防沙门氏菌感染。由于猪是沙门氏菌的重要储存库，LDLR-/-模型将具有农业和生物医学应用的双重用途。 公共卫生相关性：本建议的目的是利用从猪囊胚内细胞团(ICM)中提取的诱导多能干细胞(IPSC)系，通过核移植，即克隆，程序来生产转基因猪。因此，这些细胞很可能有相当大的潜力来操纵猪的遗传学，因此对畜牧业和生物医学科学都有价值。