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: 8618911
• 负责人: R. MICHAEL ROBERTS
• 金额: $ 29.89万
• 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/8618911
• 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.

描述（由申请人提供）：此处的目标是利用源自猪囊胚内细胞团（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，迄今为止产生的 ICM 衍生的 iPSC 依赖于 LIF，具有短的细胞周期间隔，并且类似于小鼠胚胎干细胞 (ESC)。它们可以在多次传代中存活而不会衰老，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且“未分化”的，并且可能携带它们起源的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产生更少的异常。因此，这些细胞可能具有操纵猪遗传学的巨大潜力，因此对畜牧业和一般生物医学研究有价值。目标 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，建立额外的 LIF 依赖性 ICM 衍生猪 (p) iPSC (pICM-iPSC) 系，该载体具有有效转基因沉默的优势或去除，并进一步详细描述所得线条的特征。然后将测试生长旺盛并表现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生产的能力。这种嵌合体也可能作为克隆的替代品来创造转基因猪。目标 2 是确定我们所掌握的各种 LIF 和 FGF2 依赖性 piPSC 是否是比体细胞更好的核移植供体。这一目标还将使我们能够测试代表不同类别多能性（LIF 和 FGF 依赖性）的 piPSC 之间以及 pICM-iPSC 和从体细胞重编程的类似 LIF 依赖性 piPSC 之间是否存在任何差异。一个子目标是验证 pICM-iPSC 产生的后代与核移植体细胞相比是否表现出更少的发育异常。目标 3 是建立 pICM-iPSC 中有效基因靶向和转基因的参数。将评估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转染程序和选择剂和靶向载体的有效浓度范围。目标 4 是删除 LDLR 的两个等位基因，从而通过 NT 生产出对农业和医学具有潜在兴趣的转基因猪。人类 LDLR 功能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。缺乏一个或两个拷贝的猪将为小鼠提供另一种模型，用于研究治疗这些疾病时对饮食和药物的反应。 LDLR 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宿主，LDLR-/-模型将具有农业和生物医学应用的双重目的。