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）：这里的目的是利用猪胚泡内部细胞质量（ICM）得出的诱导多能干细胞（IPSC），通过核转移（NT）程序来克隆转基因猪的克隆。在我们的实验室中，ICM衍生的IPSC到目前为止生成的IPSC是LIF依赖性的，具有短的细胞周期间隔，并且类似于小鼠胚胎干细胞（ESC）。它们可以在不屈服的情况下生存多个段落，因此适合在扩展药理学选择下引入多种遗传变化。由于细胞是多能的和“未分化的”，并且可能带有其起源的ICM的表观遗传“记忆”，因此在克隆期间，它们可能比其他体细胞类型更容易重编程，并且在诞生的Offspring中提供的异常较少。因此，这些细胞可能具有很大的潜力来操纵猪遗传学，因此对畜牧业和一般的生物医学研究具有价值。 AIM 1是通过上调在“ Tet-On” Bicistronic慢病毒载体上组装的人类POU5F1和KLF4转基因来建立其他依赖LIF依赖的ICM衍生猪（P）IPSC（PICM-IPSC）线，这提供了有效的TransCene Silencing或Removal或进一步详细介绍的，并提供了有效的TransCene Silencing或Removal和进一步的细节。然后，将测试剧烈生长并表现出全体体外和体内（畸胎瘤）多能性标准的线条，以便测试其参与嵌合体产生的能力。这种嵌合体也可能是克隆产生转基因猪的替代方案。 AIM 2是确定我们可以使用的各种LIF和FGF2依赖性PIPSC是否比核转移的体细胞比体细胞优越。这个目标还将使我们能够测试代表不同类别的多能性（LIF和FGF依赖性）的PIPSC以及PICM-IPSC和类似的LIF依赖性PIPSC之间从体细胞重新编程的。子-IAM将是验证PICM-IPSC出生的后代是否通过核转移与体细胞相比显示出更少的发育异常。 AIM 3是建立PICM-IPSC中有效基因靶向和转基因的参数。将评估转染程序和选择剂的有效浓度范围和用于引入通过同源重组编码低密度脂蛋白（LDLR）基因的一个等位基因的敲除敲除基因的敲除。 AIM 4是删除LDLR的两个等位基因，从而由NT产生具有对农业和药物潜在兴趣的转基因猪。 LDLR在人类中的功能丧失会导致高胆固醇血症，冠心病和所谓代谢综合征的发作。缺乏一个或两份副本的猪将为小鼠提供一种替代模型，以研究对这些疾病的治疗中对饮食和药物的反应。还假定LDLR缺乏症和相关的高胆固醇血症可预防沙门氏菌感染。由于猪是沙门氏菌的重要储层，因此LDLR - / - 模型将在农业和生物医学应用中具有双重目的。