Corkins Mark E, Hanania Hannah L, Krneta-Stankic Vanja, DeLay Bridget D, Pearl Esther J, Lee Moonsup, Ji Hong, Davidson Alan J, Horb Marko E, Miller Rachel K
Department of Pediatrics, Pediatric Research Center, UTHealth McGovern Medical School, Houston, TX 77030, USA.
Program in Biochemistry and Cell Biology, Rice University, Houston, TX 77005, USA.
Genes (Basel). 2018 Apr 6;9(4):197. doi: 10.3390/genes9040197.
embryos are an established model for studying kidney development. The nephron structure and genetic pathways that regulate nephrogenesis are conserved between and humans, allowing for the study of human disease-causing genes. embryos are also amenable to large-scale screening, but studies of kidney disease-related genes have been impeded because assessment of kidney development has largely been limited to examining fixed embryos. To overcome this problem, we have generated a transgenic line that labels the kidney. We characterize this :eGFP line, showing green fluorescent protein (GFP) expression in the pronephric and mesonephric kidneys and colocalization with known kidney markers. We also demonstrate the feasibility of live imaging of embryonic kidney development and the use of :eGFP as a kidney marker for secretion assays. Additionally, we develop a new methodology to isolate and identify kidney cells for primary culture. We also use morpholino knockdown of essential kidney development genes to establish that GFP expression enables observation of phenotypes, previously only described in fixed embryos. Taken together, this transgenic line will enable primary kidney cell culture and live imaging of pronephric and mesonephric kidney development. It will also provide a simple means for high-throughput screening of putative human kidney disease-causing genes.
胚胎是研究肾脏发育的成熟模型。调节肾发生的肾单位结构和遗传途径在[物种名称未给出]和人类之间是保守的，这使得能够研究人类致病基因。[物种名称未给出]胚胎也适合大规模筛选，但由于对肾脏发育的评估在很大程度上仅限于检查固定胚胎，与肾脏疾病相关基因的研究受到了阻碍。为了克服这个问题，我们构建了一个标记肾脏的转基因品系。我们对这个:eGFP品系进行了表征，显示绿色荧光蛋白（GFP）在前肾和中肾中表达，并与已知的肾脏标记物共定位。我们还证明了胚胎肾脏发育活体成像的可行性以及:eGFP作为肾脏标记物用于分泌测定的用途。此外，我们开发了一种新方法来分离和鉴定用于原代培养的肾脏细胞。我们还使用吗啉代寡核苷酸敲低关键的肾脏发育基因，以证实GFP表达能够观察到以前仅在固定胚胎中描述过的表型。综上所述，这个转基因品系将实现原代肾细胞培养以及前肾和中肾发育的活体成像。它还将为高通量筛选假定的人类肾脏致病基因提供一种简单方法。
