Abstract:
Malic Enzyme 1 (ME1) plays an integral role in fatty acid synthesis and cellular energetics through its production of NADPH and pyruvate. As such, it has been identified as a gene of interest in obesity, type 2 diabetes, and an array of epithelial cancers, with most work being performed in vitro. The current standard model for ME1 loss in vivo is the spontaneous Mod-1 null allele, which produces a canonically inactive form of ME1. Herein, we describe two new genetically engineered mouse models exhibiting ME1 loss at dynamic timepoints. Using murine embryonic stem cells and Flp/FRT and Cre/loxP class switch recombination, we established a germline Me1 knockout model (Me1 KO) and an inducible conditional knockout model (Me1 cKO), activated upon tamoxifen treatment in adulthood. Collectively, neither the Me1 KO nor Me1 cKO models exhibited deleterious phenotype under standard laboratory conditions. Knockout of ME1 was validated by immunohistochemistry and genotype confirmed by PCR. Transmission patterns favor Me1 loss in Me1 KO mice when maternally transmitted to male progeny. Hematological examination of these models through complete blood count and serum chemistry panels revealed no discrepancy with their wild-type counterparts. Orthotopic pancreatic tumors in Me1 cKO mice grow similarly to Me1 expressing mice. Similarly, no behavioral phenotype was observed in Me1 cKO mice when aged for 52 weeks. Histological analysis of several tissues revealed no pathological phenotype. These models provide a more modern approach to ME1 knockout in vivo while opening the door for further study into the role of ME1 loss under more biologically relevant, stressful conditions.
摘要:
苹果酸酶1(ME1)通过其NADPH和丙酮酸盐的产生在脂肪酸合成和细胞能量学中起着不可或缺的作用。因此,它已被确定为肥胖的基因,2型糖尿病,和一系列上皮癌,大部分工作都是在体外进行的。目前体内ME1丢失的标准模型是自发的Mod-1无效等位基因,这产生了典型的非活性形式的ME1。在这里,我们描述了两种新的基因工程小鼠模型,在动态时间点表现出ME1损失。利用鼠胚胎干细胞与Flp/FRT和Cre/loxP类开关重组,我们建立了生殖系Me1基因敲除模型(Me1KO)和诱导型条件敲除模型(Me1cKO),在成年期他莫昔芬治疗后激活。总的来说,在标准实验室条件下,Me1KO和Me1cKO模型均未表现出有害表型。通过免疫组织化学验证ME1的基因敲除,并通过PCR确认基因型。当母体传播给雄性后代时,传播模式有利于Me1KO小鼠中Me1的丢失。通过全血细胞计数和血清化学面板对这些模型进行的血液学检查显示与野生型对应物没有差异。Me1cKO小鼠中的原位胰腺肿瘤与表达Me1的小鼠相似地生长。同样,在52周龄的Me1cKO小鼠中未观察到行为表型。几种组织的组织学分析显示无病理表型。这些模型为体内ME1敲除提供了一种更现代的方法,同时为进一步研究ME1丢失在更生物学相关的情况下的作用打开了大门。紧张的条件。