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（由申请人提供）：本文的目标是利用来自猪囊胚内细胞团（ICM）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，到目前为止产生的ICM衍生的iPSC是LIF依赖的，具有短的细胞周期间隔，类似于小鼠胚胎干细胞（ESC）。它们可以在多次传代后存活而不衰老，因此适合在扩展的药理学选择下引入多种遗传变化。由于这些细胞是多能性的和“未分化的”，并且可能携带着它们所起源的ICM的表观遗传“记忆”，因此它们在克隆过程中可能比其他体细胞类型更容易重新编程，并且在出生的后代中提供更少的异常。因此，这些细胞可能具有相当大的潜力，用于操纵猪遗传学，因此对畜牧业和生物医学研究具有价值。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POU 5 F1和KLF 4转基因来建立LIF依赖性ICM衍生的猪（p）iPSC（pICM-iPSC）的额外系，其提供有效转基因沉默或去除的优点，并进一步详细表征所得系。然后将测试生长旺盛并显示出完整的体外和体内（畸胎瘤）多能性标准的细胞系参与嵌合体产生的能力。这种嵌合体也可能是有价值的替代克隆创造转基因猪。目的2是确定在我们处置下的各种LIF和FGF 2依赖性piPSC是否是比体细胞上级的用于核转移的供体。该目的还将允许我们测试在代表不同类别的多能性（LIF依赖性和FGF依赖性）的piPSC之间以及在pICM-iPSC和从体细胞重编程的类似的LIF依赖性piPSC之间是否存在任何差异。子目标将是验证与通过核移植的体细胞相比，pICM-iPSC出生的后代是否显示出更少的发育异常。目的3是建立pICM-iPSC中有效基因靶向和转基因的参数。将评价用于通过同源重组引入编码低密度脂蛋白（LDLR）受体的基因的等位基因之一敲除的选择剂和靶向载体的转染程序和有效浓度范围。目的4是通过NT技术使LDLR的两个等位基因缺失，从而获得具有农业和医学双重价值的转基因猪。人体中LDLR功能的丧失导致高胆固醇血症、冠心病和所谓的代谢综合征的发作。缺乏一个或两个拷贝的猪将为小鼠提供另一种模型，用于研究在治疗这些疾病时对饮食和药物的反应。LDLR缺乏症和相关的高胆固醇血症也被认为可以防止沙门氏菌感染。由于猪是沙门氏菌的重要宿主，LDLR-/-模型将具有农业和生物医学应用的双重目的。 公共卫生关系：本提案的目的是利用源自猪囊胚内细胞团（ICM）的诱导多能干细胞（iPSC）系，通过核转移（即克隆）程序生产转基因猪。因此，这些细胞可能具有相当大的潜力，操纵猪的遗传学，因此是有价值的畜牧业和生物医学科学。