Gene imprinting and obesity, a new pig model

描述（由申请人提供）：Prader-Willi综合征（PWS）是一种多系统疾病，由约12个父亲表达的印迹基因簇功能丧失引起。PWS的主要特征包括新生儿发育不全、身体组成异常、身材矮小伴生长激素缺乏、儿童期暴饮暴食和肥胖，以及其他内分泌和行为异常。尽管PWS的小鼠模型概括了该疾病的一些临床成分，但它们不会发展为早发性贪食或人类疾病的严重肥胖。因此，需要替代动物模型来研究该疾病多变性的临床特征，特别是饮食失调和肥胖的生物医学基础和治疗方法。我们认为小型猪（minipigs）是理想的PWS模型。在迷你猪中存在产生疾病遗传模型的技术。最重要的是，它们的体型、生理、解剖结构和基因组都比老鼠更接近人类。因此，我们假设患有PWS的迷你猪比相应的小鼠模型更容易发生肥胖。我们最近绘制了猪PWS-同源区域的克隆图谱和DNA序列，确定了控制印迹的主要调控元件和PWS-区域的所有印迹基因。因此，基因组工具现在已经到位，可以产生猪PWS模型。本应用程序的具体目的1是在雄性和雌性胎猪细胞中使用TALEN（转录激活因子样效应核酸酶）方法来靶向母体等位基因上印迹控制（IC）区域的缺失。具体目标2是利用体细胞核移植培育动物。针对雄性细胞和父系传播将很快提供pws模型，从而确认猪模型在研究贪食和肥胖方面的实用性；然而，这些猪可能不育，就像人类PWS的情况一样。通过靶向雌性细胞中的沉默母系等位基因，我们将能够在正常的雌性载体猪系中维持突变，只有在父系传播突变后，才能生产出PWS和野生型对照仔猪，用于临床、病理生理和分子研究。猪PWS模型将有助于了解PWS的贪食和肥胖，并为治疗试验提供手段。此外，这项工作将为确定PWS的生物学途径如何与人类肥胖的遗传、饮食和行为原因以及体重稳态的已知成分相关奠定基础。

DESCRIPTION (provided by applicant): Prader-Willi syndrome (PWS) is a multisystem disorder caused by loss of function of a cluster of ~12 paternally-expressed, imprinted genes. Cardinal features of PWS include neonatal failure to thrive, abnormal body composition, short stature with growth hormone deficiency, and childhood-onset hyperphagia and obesity, among other endocrine and behavioral abnormalities. Although mouse models of PWS recapitulate some of the clinical components of the disorder, they do not develop early- onset hyperphagia or the severe obesity of the human disease. Therefore, alternative animal models are needed to study the biomedical basis and therapeutic approaches for the pleiotropic clinical features of the disorder, especially the eating disorder and obesity. We propose that miniature pigs (minipigs) are ideal to generate a model for PWS. Technologies exist in the minipig to produce genetic models of disease. Most importantly, they have a more similar body size, physiology, anatomy, and genome to human than does the mouse. As a result, we hypothesize that minipigs with PWS will be more susceptible to development of obesity than the corresponding mouse models. We have recently generated a clone map and the DNA sequence for the pig PWS-orthologous region, defining the major regulatory elements that control imprinting and all the imprinted genes in the PWS- domain. Thus, the genomic tools are now in place to produce a porcine model of PWS. Specific Aim 1 of this application is to use a TALEN (Transcription Activator-Like Effector Nuclease) approach in male and female fetal pig cells to target a deletion of the imprinting control (IC) region on the maternal allele. Specific Aim 2 is to use somatic cell nuclear transfer to generate animals. Targeting in male cells and paternal transmission will quickly provide a PWS-model, allowing confirmation of the utility of a pig model to study hyperphagia and obesity; however, these pigs may be infertile as is the case in human PWS. By targeting the silent maternal allele in female cells, we will be able to maintain the mutation in a normal, female carrier pig line that only after paternal transmission of the mutation will produce litters of PWS and wildtype control piglets for clinical, pathophysiological, and molecular studies. A porcine model of PWS will lead to an understanding of hyperphagia and obesity in PWS and provide a means for therapeutic testing. Additionally, this work will set the stage to determine how biological pathways in PWS relate to genetic, dietary, and behavioral causes of human obesity and known components of body weight homeostasis.