Induced Pluripotent Stem Cells from Swine: application to genetic modification

描述（由申请人提供）：这里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，迄今为止生成的icm衍生的iPSC依赖于liff，具有较短的细胞周期间隔，类似于小鼠胚胎干细胞（ESC）。它们可以在多次传代中存活而不衰老，因此适合在扩展的药物选择下引入多种遗传变化。由于这些细胞是多能和“未分化”的，并且可能携带它们起源的ICM的表观遗传“记忆”，它们可能比其他体细胞类型更容易在克隆过程中重新编程，并且在出生的后代中提供更少的异常。因此，这些细胞很可能具有操纵猪遗传的巨大潜力，因此对畜牧业和一般的生物医学研究具有价值。目的1是通过上调组装在双胞慢病毒载体上的人POU5F1和KLF4转基因，建立更多的依赖于lifm的icm衍生的猪(p) iPSC （pICM-iPSC）细胞系，这提供了有效的转基因沉默或去除的优势，并进一步详细地描述了所产生的细胞系。在体外和体内（畸胎瘤）中表现出充分的多能性标准的细胞系将被测试其参与嵌合体生产的能力。这种嵌合体也可能是一种有价值的替代克隆，以创造转基因猪。目的2是确定我们所掌握的各种依赖于lif2和fgf2的piPSC是否比体细胞更适合核移植。这一目标也将允许我们测试代表不同类型多能性（liff和fgf依赖）的piPSC之间，以及从体细胞重编程的pICM-iPSC和类似的liff依赖的piPSC之间是否存在差异。一个次要目标将是验证pICM-iPSC所生的后代是否比通过核移植的体细胞表现出更少的发育异常。目的3是建立pICM-iPSC中有效的基因靶向和转基因参数。将评估转染程序和选择剂和靶向载体的有效浓度范围，用于通过同源重组引入编码低密度脂蛋白（LDLR）受体基因的一个等位基因的敲除。目标4是通过NT删除LDLR的两个等位基因，从而产生对农业和医学都有潜在兴趣的转基因猪。人类LDLR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作。缺乏一个或两个副本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储存库，LDLR-/-模型将具有农业和生物医学应用的双重目的。

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.