Eshbach Megan L, Sethi Rahil, Avula Raghunandan, Lamb Janette, Hollingshead Deborah J, Finegold David N, Locker Joseph D, Chandran Uma R, Weisz Ora A
Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
Department of Biomedical Informatics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
Am J Physiol Renal Physiol. 2017 Sep 1;313(3):F585-F595. doi: 10.1152/ajprenal.00228.2017. Epub 2017 Jun 14.
The OK cell line derived from the kidney of a female opossum has proven to be a useful model in which to investigate the unique regulation of ion transport and membrane trafficking mechanisms in the proximal tubule (PT). Sequence data and comparison of the transcriptome of this cell line to eutherian mammal PTs would further broaden the utility of this culture model. However, the genomic sequence for is not available and although a draft genome sequence for the opossum (sequenced in 2012 by the Broad Institute) exists, transcripts sequenced from both species show significant divergence. The sequence is not highly annotated, and the majority of transcripts are predicted rather than experimentally validated. Using deep RNA sequencing of the OK cell line, we characterized its transcriptome via de novo transcriptome assembly and alignment to the genome. The quality of the de novo assembled transcriptome was assessed by the extent of homology to sequences in nucleotide and protein databases. Gene expression levels in the OK cell line, from both the de novo transcriptome and genes aligned to the genome, were compared with publicly available rat kidney nephron segment expression data. Our studies demonstrate the expression in OK cells of numerous PT-specific ion transporters and other key proteins relevant for rodent and human PT function. Additionally, the sequence and expression data reported here provide an important resource for genetic manipulation and other studies on PT cell function using these cells.
源自雌性负鼠肾脏的OK细胞系已被证明是一种有用的模型，可用于研究近端小管（PT）中离子转运和膜运输机制的独特调节。该细胞系的序列数据以及其转录组与真兽类哺乳动物近端小管的比较，将进一步扩大这种培养模型的用途。然而，目前尚无该物种的基因组序列，尽管存在负鼠的基因组草图序列（由布罗德研究所于2012年测序），但两个物种的转录本测序显示出显著差异。该物种的序列注释不充分，大多数转录本是预测的而非经过实验验证的。通过对OK细胞系进行深度RNA测序，我们通过从头转录组组装并与该物种基因组比对来表征其转录组。通过与核苷酸和蛋白质数据库中序列的同源程度评估从头组装转录组的质量。将从头转录组和与该物种基因组比对的基因在OK细胞系中的基因表达水平，与公开可用的大鼠肾脏肾单位节段表达数据进行比较。我们的研究证明了OK细胞中许多近端小管特异性离子转运蛋白以及与啮齿动物和人类近端小管功能相关的其他关键蛋白的表达。此外，本文报道的序列和表达数据为利用这些细胞进行近端小管细胞功能的基因操作和其他研究提供了重要资源。
