Nemtsova Yuliya, Wiseman Jennifer A, El-Banna Mukarram, Lobel Peter, Sleat David E
Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey, United States of America.
Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers Biomedical Health Sciences, Rutgers University, Piscataway, New Jersey, United States of America.
PLoS One. 2018 Feb 6;13(2):e0192286. doi: 10.1371/journal.pone.0192286. eCollection 2018.
Late-infantile neuronal ceroid lipofuscinosis is a fatal neurodegenerative disease of children caused by mutations resulting in loss of activity of the lysosomal protease, tripeptidyl peptidase 1 (TPP1). While Tpp1-targeted mouse models of LINCL exist, the goal of this study was to create a transgenic mouse with inducible TPP1 to benchmark treatment approaches, evaluate efficacy of treatment at different stages of disease, and to provide insights into the pathobiology of disease. A construct containing a loxP-flanked stop cassette inserted between the chicken-actin promoter and a sequence encoding murine TPP1 (TgLSL-TPP1) was integrated into the ROSA26 locus in mice by homologous recombination. Tested in both transfected CHO cells and in transgenic mice, the TgLSL-TPP1 did not express TPP1 until cre-mediated removal of the LSL cassette, which resulted in supraphysiological levels of TPP1 activity. We tested four cre/ERT2 transgenes to allow tamoxifen-inducible removal of the LSL cassette and subsequent TPP1 expression at any stage of disease. However, two of the cre/ERT2 driver transgenes had significant cre activity in the absence of tamoxifen, while cre-mediated recombination could not be induced by tamoxifen by two others. These results highlight potential problems with the use of cre/ERT2 transgenes in applications that are sensitive to low levels of basal cre expression. However, the germline-recombined mouse transgenic that constitutively overexpresses TPP1 will allow long-term evaluation of overexposure to the enzyme and in cell culture, the inducible transgene may be a useful tool in biomarker discovery projects.
晚期婴儿神经元蜡样脂褐质沉积症是一种儿童致命性神经退行性疾病,由导致溶酶体蛋白酶三肽基肽酶1(TPP1)活性丧失的突变引起。虽然存在针对LINCL的Tpp1基因靶向小鼠模型,但本研究的目的是创建一种具有可诱导TPP1的转基因小鼠,以确定治疗方法的基准,评估疾病不同阶段的治疗效果,并深入了解疾病的病理生物学。通过同源重组将一个构建体整合到小鼠的ROSA26位点,该构建体包含一个插入鸡肌动蛋白启动子和编码小鼠TPP1序列(TgLSL-TPP1)之间的loxP侧翼终止盒。在转染的CHO细胞和转基因小鼠中进行测试,TgLSL-TPP1在cre介导去除LSL盒之前不表达TPP1,这导致TPP1活性超生理水平。我们测试了四个cre/ERT2转基因,以允许他莫昔芬诱导去除LSL盒并在疾病的任何阶段随后表达TPP1。然而,其中两个cre/ERT2驱动转基因在没有他莫昔芬的情况下具有显著的cre活性,而另外两个则不能通过他莫昔芬诱导cre介导的重组。这些结果突出了在对低水平基础cre表达敏感的应用中使用cre/ERT2转基因的潜在问题。然而,组成型过表达TPP1的种系重组小鼠转基因将允许对酶的过度暴露进行长期评估,并且在细胞培养中,可诱导转基因可能是生物标志物发现项目中的有用工具。