Automated method for the isolation of collecting ducts.
Automated method for the isolation of collecting ducts.
复制标题
隔离集合管的自动化方法。
DOI：
10.1152/ajprenal.00273.2005
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Nelson,RaoulD
中科院分区：
文献类型：
--
作者：
Miller,RLance;Zhang,Ping;Chen,Tong;Rohrwasser,Andreas;Nelson,RaoulD
收集管的结构和功能的异质性提出了一个巨大的实验挑战，需要手动显微解剖，这是耗时、劳动密集型的，而且不适合高通量。为了克服这些局限性，我们开发了一种新的方法，将在集合管中表达绿色荧光蛋白(GFP)的转基因小鼠与基于大颗粒的流式细胞术相结合，从整个集合管(CD)或内髓(IMCD)、外髓(OMCD)或连接段/皮质集合管(CNT/CCD)中分离纯净的小管片段。肾脏被酶分散成管状碎片，并使用基于大颗粒的流式细胞仪或复杂对象参数分析仪和分类器(COPAS)根据管长和GFP强度进行分类。活体/死亡实验表明，这些小管90%存活。收集小管作为荧光强度的函数，并通过荧光和位相显微镜分析计数准确性、GFP阳性、平均小管长度和收集100个小管所需的时间。类似地，利用实时定量RT-PCR和免疫印迹技术分析分离的小管中的mRNA和蛋白表达的小管段特异性基因。分选后的小管纯度和得率与分选的严密性有关。每只小鼠在1h内常规获得100条收集管(9.68±0.44~14.5±0.66 cm或9.2±0.7 mg管状蛋白)的4~6次重复，表明大颗粒流式细胞术快速、重复性好，可以从单个或重复动物身上获得足够数量的高纯度和可存活的收集管，用于基因表达和蛋白质组学分析。
The structural and functional heterogeneity of the collecting duct present a tremendous experimental challenge requiring manual microdissection, which is time-consuming, labor intensive, and not amenable to high throughput. To overcome these limitations, we developed a novel approach combining the use of transgenic mice expressing green fluorescent protein (GFP) in the collecting duct with large-particle-based flow cytometry to isolate pure populations of tubular fragments from the whole collecting duct (CD), or inner medullary (IMCD), outer medullary (OMCD), or connecting segment/cortical collecting duct (CNT/CCD). Kidneys were enzymatically dispersed into tubular fragments and sorted based on tubular length and GFP intensity using large-particle-based flow cytometry or a complex object parametric analyzer and sorter (COPAS). A LIVE/DEAD assay demonstrates that the tubules were >90% viable. Tubules were collected as a function of fluorescent intensity and analyzed by epifluorescence and phase microscopy for count accuracy, GFP positivity, average tubule length, and time required to collect 100 tubules. Similarly, mRNA and protein from sorted tubules were analyzed for expression of tubule segment-specific genes using quantitative real-time RT-PCR and immunoblotting. The purity and yield of sorted tubules were related to sort stringency. Four to six replicates of 100 collecting ducts (9.68 ± 0.44–14.5 ± 0.66 cm or 9.2 ± 0.7 mg tubular protein) were routinely obtained from a single mouse in under 1 h. In conclusion, large-particle-based flow cytometry is fast, reproducible, and generates sufficient amounts of highly pure and viable collecting ducts from single or replicate animals for gene expression and proteomic analysis.
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体内肾小球特异性基因切除。
影响因子：
13.6
作者：
V. Eremina;M. Wong;S. Cui;L. Schwartz;S. Quaggin
通讯作者：
S. Quaggin
小鼠 THP 基因启动子的分离及其在转基因小鼠中的肾脏特异性活性的证明。
DOI：
10.1152/ajprenal.00297.2001
发表时间：
2002
期刊：
American journal of physiology. Renal physiology
影响因子：
--
作者：
Zhu,Xinhua;Cheng,Jin;Gao,Jing;Lepor,Herbert;Zhang,Zhong-Ting;Pak,Joanne;Wu,Xue-Ru
通讯作者：
Wu,Xue-Ru
对照组和氯化铵负载兔集合管中带 3 阳性嵌入细胞的轴向分布。
DOI：
--
发表时间：
1996
期刊：
Kidney international. Supplement.
影响因子：
--
作者：
Verlander,JW;Madsen,KM;Tisher,CC
通讯作者：
Tisher,CC
小鼠连接小管中的肾素和激肽释放酶。
DOI：
10.1046/j.1523-1755.2003.00302.x
发表时间：
2003
期刊：
Kidney international.
影响因子：
--
作者：
Rohrwasser,Andreas;Ishigami,Tomoaki;Gociman,Barbu;Lantelme,Pierre;Morgan,Terry;Cheng,Tong;Hillas,Elaine;Zhang,Shuhua;Ward,Kenneth;Bloch-Faure,May;Meneton,Pierre;Lalouel,JM
通讯作者：
Lalouel,JM
DOI：
10.1152/ajprenal.1995.269.5.f663
发表时间：
1995-11-01
期刊：
AMERICAN JOURNAL OF PHYSIOLOGY-RENAL FLUID AND ELECTROLYTE PHYSIOLOGY
影响因子：
--
作者：
ECELBARGER, CA;TERRIS, J;KNEPPER, MA
通讯作者：
KNEPPER, MA