Vanderbilt Jeff N, Allen Lennell, Gonzalez Robert F, Tigue Zachary, Edmondson Jess, Ansaldi Daniel, Gillespie Anne Marie, Dobbs Leland G
Cardiovascular Research Institute, University of California San Francisco, San Francisco, California 94118, USA.
Am J Respir Cell Mol Biol. 2008 Sep;39(3):253-62. doi: 10.1165/rcmb.2008-0049OC. Epub 2008 Mar 26.
Podoplanin (RTI40, aggrus, T1alpha, hT1alpha-2, E11, PA2.26, RANDAM-2, gp36, gp38, gp40, OTS8) is a type I cell marker in rat lung. We show that a bacterial artificial chromosome vector containing the rat podoplanin gene (RTIbac) delivers a pattern of transgene expression in lung that is more restricted to mouse type I cells than that of the endogenous mouse podoplanin gene. RTIbac-transgenic mice expressed rat podoplanin in type I cells; type II cells, airways, and vascular endothelium were negative. A modified bacterial artificial chromosome containing internal ribosome entry site (IRES)-green fluorescent protein (GFP) sequences in the podoplanin 3'UTR expressed rat podoplanin and transgenic GFP in type I cells. RTIbac transgene expression was absent or reduced in pulmonary pleura, lymphatic endothelium, and putative lymphoid-associated stromal tissue, all of which contained abundant mouse podoplanin. Rat podoplanin mRNA levels in normal rat lung and RTIbac transgenic lung were 25-fold higher than in corresponding kidney and brain samples. On Western blots, transgenic rat and endogenous mouse podoplanin displayed very similar patterns of protein expression in various organs. Highest protein levels were observed in lung with 10- to 20-fold less in brain; there were low levels in thymus and kidney. Both GFP and rat podoplanin transgenes were expressed at extrapulmonary sites of endogenous mouse podoplanin gene expression, including choroid plexus, eye ciliary epithelium, and renal glomerulus. Because their pulmonary expression is more restricted than endogenous mouse podoplanin, RTIbac derivatives should be useful for mouse type I cell-specific transgene delivery.
血小板反应蛋白-1结构域蛋白（RTI40、聚集蛋白、T1α、hT1α-2、E11、PA2.26、RANDAM-2、gp36、gp38、gp40、OTS8）是大鼠肺中的I型细胞标志物。我们发现，含有大鼠血小板反应蛋白-1结构域蛋白基因的细菌人工染色体载体（RTIbac）在肺中产生的转基因表达模式比内源性小鼠血小板反应蛋白-1结构域蛋白基因更局限于小鼠I型细胞。RTIbac转基因小鼠在I型细胞中表达大鼠血小板反应蛋白-1结构域蛋白；II型细胞、气道和血管内皮均为阴性。一种在血小板反应蛋白-1结构域蛋白3'非翻译区含有内部核糖体进入位点（IRES）-绿色荧光蛋白（GFP）序列的修饰细菌人工染色体在I型细胞中表达大鼠血小板反应蛋白-1结构域蛋白和转基因GFP。在肺胸膜、淋巴管内皮和假定的淋巴相关基质组织中，RTIbac转基因表达缺失或减少，所有这些组织都含有丰富的小鼠血小板反应蛋白-1结构域蛋白。正常大鼠肺和RTIbac转基因肺中的大鼠血小板反应蛋白-1结构域蛋白mRNA水平比相应的肾脏和脑样本高25倍。在蛋白质印迹上，转基因大鼠和内源性小鼠血小板反应蛋白-1结构域蛋白在各种器官中显示出非常相似的蛋白质表达模式。在肺中观察到最高的蛋白质水平，在脑中则低10至20倍；在胸腺和肾脏中水平较低。GFP和大鼠血小板反应蛋白-1结构域蛋白转基因均在内源性小鼠血小板反应蛋白-1结构域蛋白基因表达的肺外部位表达，包括脉络丛、眼睫状体上皮和肾小球。由于它们在肺中的表达比内源性小鼠血小板反应蛋白-1结构域蛋白更局限，RTIbac衍生物应可用于小鼠I型细胞特异性转基因递送。
