Department of Ophthalmology, Jacobs School of Medicine and Biomedical Sciences, The State University of New York - University at Buffalo, Buffalo, NY, USA; Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, The State University of New York - University at Buffalo, Buffalo, NY, USA; Neuroscience Graduate Program, Jacobs School of Medicine and Biomedical Sciences, The State University of New York - University at Buffalo, Buffalo, NY, USA; Research Service, VA Western New York Healthcare System, Buffalo, NY, USA.
Department of Ophthalmology, Jacobs School of Medicine and Biomedical Sciences, The State University of New York - University at Buffalo, Buffalo, NY, USA; Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, The State University of New York - University at Buffalo, Buffalo, NY, USA; Neuroscience Graduate Program, Jacobs School of Medicine and Biomedical Sciences, The State University of New York - University at Buffalo, Buffalo, NY, USA; Research Service, VA Western New York Healthcare System, Buffalo, NY, USA.
Exp Eye Res. 2023 Oct;235:109637. doi: 10.1016/j.exer.2023.109637. Epub 2023 Aug 31.
Although cell type-specific Cre recombinase-expressing mouse lines are commonly used to generate conditional knockout of genes of interest, germline recombination and ectopic "leakiness" in Cre recombinase expression in non-specific cell types has been observed in several neuronal and glial-specific Cre lines. This often leads to inadvertent loss of conditional mouse lines, requiring rederivation. It is therefore imperative to be able to monitor and validate cell type-specific Cre recombinase-mediated gene editing. Herein, we describe a simple, inexpensive, rapid ZsGreen fluor-reporter-based strategy for genotype-free identification of ectopic leakiness using a custom-designed, 3-D blue LED light box. We assessed cell type-specific expression in several allegedly specific Cre recombinase mouse lines commonly used in vision research: retinal pigment epithelium (RPE)-specific (VMD2 (Best1) Cre, RPE65 Cre); astrocyte-specific (GFAP Cre); as well as photoreceptor-bipolar progenitor cell-specific (CRX Cre). Our standardized workflow allows facile, rapid identification of ectopic and non-specific Cre recombinase expression in any presume specific Cre mouse line, without the need for genotyping and without causing animal distress.
尽管细胞类型特异性 Cre 重组酶表达的小鼠品系常用于生成目的基因的条件性敲除，但在几种神经元和神经胶质特异性 Cre 线中已经观察到生殖系重组和 Cre 重组酶在非特异性细胞类型中的异位“渗漏”。这通常会导致意外丢失条件性小鼠系，需要重新衍生。因此，能够监测和验证细胞类型特异性 Cre 重组酶介导的基因编辑是至关重要的。在这里，我们描述了一种简单、廉价、快速的基于 ZsGreen 荧光报告基因的策略，用于使用定制的 3-D 蓝色 LED 灯箱进行基因型免费的异位渗漏识别。我们评估了几种常用于视觉研究的据称特异性 Cre 重组酶小鼠线中的细胞类型特异性表达：视网膜色素上皮（RPE）特异性（VMD2（Best1）Cre、RPE65 Cre）；星形胶质细胞特异性（GFAP Cre）；以及光感受器-双极祖细胞特异性（CRX Cre）。我们的标准化工作流程允许在任何假定的特异性 Cre 小鼠系中轻松、快速地识别异位和非特异性 Cre 重组酶表达，而无需进行基因分型，也不会引起动物不适。
