异种移植血管内皮细胞组织特异性表达人CD59转基因小鼠的建立

目的建立用于异种移植研究血管内皮细胞组织特异性表达人CD59转基因小鼠。方法采用受精卵显微注射技术，将含有人ICAM-2启动子、人CD59基因第1个内含子、人CD59 cDNA、BGH polyA终止信号的外源基因导入小鼠受精卵的原核中。选取注射后仍健康的受精卵移植入假孕母鼠的输卵管中待分娩。聚合酶链反应（PCR）及Southern blot确定外源基因整合阳性转基因小鼠。流式细胞术用于外源基因蛋白质水平表达检测。免疫组织化学方法观察人CD59在转基因小鼠心脏、肝脏、肾脏等器官表达分布情况。结果产仔130只，9只外源基因整合阳性。整合率6％（9／130）。6只获得蛋白质水平表达。蛋白质水平表达强度为人CD59在人白细胞表达强度的80％至95％，转基因效率5％（6／130）。免疫组织化学检测显示人CD59在转基因小鼠心脏、肝脏、肾脏组织有较强表达，且表达限于血管内皮细胞。结论成功地建立了用于异种移植研究血管内皮细胞组织特异性表达人CD59转基因小鼠。     

包含UI的人衰变加速因子重组基因在转基因鼠高效表达的研究

目的建立血管内皮细胞高效表达人衰变加速因子（hDAF）基因的转基因小鼠。方法采用受精卵显微注射法，将包含杂合内含子（UI）的人hDAF导入昆明白小鼠受精卵的雄原核内，然后将注射后仍健康的受精卵植入假孕母鼠输卵管内，待其自然分娩。应用聚合酶链反应（PCR）和Southern杂交检测G0代小鼠hDAF基因的整合情况，应用流式细胞计数（FCM）检测小鼠外周血单核细胞（PBMCs）表面hDAF的表达，应用逆转录（RT）-PCR检测转基因小鼠心脏、肝脏、肾脏和肺脏人hDAF基因mRNA的表达。结果注射后卵的存活率和幼鼠出生率分别为83．8％（941／1123）和9．5％（89／941），外源基因的整合率为22．5％（20／89），FCM检测发现其中有7只转基因小鼠PBMCs有人hDAF表达，表达强度为人PBMCs的152％～243％，并且相应小鼠的心、肝、肾及肺组织均有人hDAFmRNA表达。结论UI可以使hDAF在转基因鼠各主要脏器高效表达。     

建立血管内皮细胞组织特异性表达人衰变加速因子的转基因小鼠

目的为了研究异种移植，建立血管内皮细胞组织特异性表达人衰变加速因子(DAF)的转基因小鼠。方法采用受精卵显微注射技术，将含有人内皮细胞粘附分子-2(ICAM-2)基因启动子、人DAF cDNA(插有人DAF基因第一个内含子)、SV40splice／polyA的外源基因导人小鼠受精卵的原核中；选取注射后仍健康的受精卵移植入假孕母鼠的输卵管中待分娩。聚合酶链(PCR)技术及Southern印迹杂交法确定外源基因整合阳性转基因小鼠。RT-PCR方法和流式细胞术分别用于外源基因mRNA及蛋白质水平表达的检测。免疫组织化学方法观察人DAF在转基因小鼠心脏、肝脏、肾脏等器官的表达分布。采用Langendorff心脏灌流装置，用200g／L的稀释人血清灌注转基因小鼠离体心脏，检测其抗超急性排斥反应能力。结果共产仔鼠133只，21只整合有外源基因，整合率16％(21／133)。8只实现mRNA及蛋白质水平表达，蛋白质水平表达强度为人DAF基因在人白细胞表达强度的70％至95％，转基因效率60A(8／133)。免疫组织化学法显示人DAF在转基因小鼠器官组织切片上有较强表达，且表达限于血管内皮细胞。与普通鼠对比，转基因小鼠离体心脏存活时间明显延长，且60min灌注期间内做功仍维持在最大值20％以上。结论成功地建立了血管内皮细胞组织特异性表达人DAF的转基因小鼠。这种小鼠有一定的抗超急性排斥反应能力。     

小鼠血管内皮细胞表达人DAF和CD59对其心脏在人血清灌注下做功的影响

目的探讨联合转人衰变加速因子（DAF）和CD59基因对小鼠心脏在人血清灌注下做功及组织结构的影响。方法采用受精卵显微注射技术，将人DAF和CD59基因注入昆明小鼠受精卵的原核中，选取注射后仍健康的受精卵植入假孕母鼠的输卵管中待分娩。提取足月产小鼠（G0代）基因组DNA，以聚合酶链反应（PCR）及Southern印迹杂交确定外源基因整合情况，应用流式细胞术检测人DAF和CD59在转基因小鼠白细胞上的表达，免疫组织化学染色检测人DAF和CD59在转基因小鼠心脏、肝脏及肾脏组织中的表达。取出转基因成功小鼠的心脏，在体外以人血清进行灌注，测定其做功能力，HE染色及免疫组化染色观察灌注后心脏的组织学变化以及补体C3c的沉积情况。以单一转DAF基因的小鼠为对照。结果共获得G0代小鼠135只，经PCR及Southern印迹杂交分析，11只（8．1%，11／135）整合有人DAF和CD59外源基因，其中6只的白细胞膜表面人DAF和CD59表达阳性，强度分别为（86±6）％和（85±8）%，单转人DAF基因者（87±7）%，差异无统计学意义。人DAF及CD59仅表达于转基因小鼠心脏、肝脏及肾脏组织中的血管内皮细胞上。在人血清灌注后60min内，普通小鼠的心脏做功急剧下降，接近40min时心脏停止搏动；转DAF基因小鼠的心脏做功也下降，但仍维持在最大值的20%以上；转DAF和CD59双基因小鼠的心脏做功维持在最大值的40％以上。在人血清灌注的10～60min，转基因小鼠心脏做功能力明显强于普通小鼠心脏（P〈0．05）；灌注20-60min时，转双基因小鼠的心脏做功能力明显强于转DAF基因小鼠心脏（P〈0．05）。普通小鼠心脏在人血清灌注后组织裂解较多见，肿胀严重，而转基因小鼠心脏组织的病理改变明显轻于普通小鼠，转双基因者的病理变化又明显轻于单一转DAF者，心肌血管中人C3c的沉积也明显少于单一转DAF者。结论血管内皮细胞特异性表达人DAF和CD59，可明显阻抑其心脏在异种血清灌注下的做功能力下降及C3c在血管中的沉积，这可能对抵御超急性排斥反应有一定帮助。     

携带人衰变加速因子基因转基因小鼠的建立

目的 通过建立转人衰变加速因子（ＤＡＦ）基因小鼠,为研究异种器官移植的超急性排斥反应提供有效的研究手段。方法 采用受精卵显微注射技术,将人ＤＡＦ基因导入小白鼠受精卵的原核中,Ｄｏｔ－ｂｌｏｔ及Ｓｏｕｔｈｅｒｎ－ｂｌｏｔ杂交确定阳性围基因小鼠。以Ｎｏｒｈｅｒｎ杂交法检测人ＤＡＦ基因的表达情况。结果 基因注射后共生出小鼠２４只,４只为转基因小鼠,人ＤＡＦ基因在其中１只小鼠体内得到表达,结论 所导入ＤＡ     

核定位信号肽在异种移植动物模型转人α1,2-岩藻糖苷转移酶基因小鼠制备中的作用

目的探讨核定位信号肽（NLS）在转人α1，2-岩藻糖苷转移酶（HT）基因小鼠制备中的作用。方法将受精卵随机分为3组，采用显微注射的方法制备转基因小鼠。实验组：将NLS转基因构件与人HT转基因构件同时导入细胞质；对照Ⅰ组：将人HT转基因构件导入细胞质；对照Ⅱ组：将人HT转基因构件导入细胞核。应用聚合酶链反应（PCR）、Southern杂交及逆转录（RT）-PCR等方法检测人HT基因在G0代小鼠体内的整合与表达，应用流式细胞计数（FCM）检测转基因小鼠外周血单核细胞（PBMCs）表面H抗原与α-Gal抗原的表达。结果实验组与对照Ⅰ组G0代小鼠的出生率33．8%（46／136）及35．4%（23／65）高于对照Ⅱ组10．2％（51／498）（P〈0．01），实验组人HT基因的整合率（30．4%，14／46）高于对照Ⅰ组（0，0／23）与对照Ⅱ组（11．8%，6／51，P〈0．01），实验组人HT基因的表达率（19．6%，9／46）也高于对照Ⅰ组（0，0／23）和对照Ⅱ组（5．9%,3／51，P〈0．01），人HT mRNA在小鼠心、肝、肾和骨骼肌组织中均表达阳性。转基因小鼠PBMCs表面H抗原表达阳性，表达强度为人的85％～190%，同时α-Gal抗原表达显著降低。为正常小鼠的5%～12％。转基因小鼠已经稳定传3代。结论NLS基因与目的基因细胞质共注射是制备转基因小鼠简便实用的新方法。     

Spyda基因过表达转基因小鼠品系的建立

目的建立Spyda基因过表达转基因小鼠品系用于深入研究Spyda基因的功能。方法利用Gateway技术构建Spyda表达载体,通过DNA显微注射的方式获得Spyda基因过表达的首建鼠。首建鼠与野生型鼠杂交,所得子代中的阳性鼠同窝交配,已传3代以后的阳性小鼠经SYBR Green实时荧光定量PCR方法检测外源基因的起始模板量,通过与杂合子比较来预测小鼠的基因型,再用传统育种方式验证SYBR Green实时荧光定量PCR的结果。结果获得7只首建鼠,通过与野生型鼠杂交获得阳性子代,进行同窝交配,传至第3代,经实时荧光定量PCR方法筛选出4只纯合子的小鼠,经测交验证,其与正常小鼠交配所生的后代均为阳性,证明实时定量PCR的结果是正确的。建立了2个独立的Spyda转基因小鼠纯合子品系。结论建立了稳定遗传的纯合子Spyda基因过表达转基因小鼠品系,可作为工具鼠进一步深入研究Spyda基因的功能。     

Gpx1-Klk1转基因对肾缺血再灌注损伤保护作用的研究

目的 制备人谷胱甘肽过氧化物酶（Gpx1）、 组织激肽释放酶（Klk1）共转基因小鼠,在此基础上制作肾缺血再灌注损伤小鼠模型。观察转基因小鼠对缺血再灌注损伤的耐受能力。在体研究Gpx1-Klk1转基因对肾缺血再灌注的保护作用。 方法 应用基因工程技术制备pKsp-Gpx1-IRES-Klk1质粒,酶切后回收转基因片段并纯化,通过外源基因受精卵雄原核显微注射法制备Gpx1-Klk1共转基因小鼠。采用丝线悬吊控制法制备转基因小鼠肾缺血再灌注模型。设立C57BL野生型小鼠同法制备的肾缺血再灌注损伤为对照。在肾缺血再灌注实验前后,测定血尿素氮、血肌酐、肾组织中超氧化物歧化酶（SOD）、丙二醛（MDA）、总一氧化氮合酶（tNOS）及诱导型一氧化氮合酶（iNOS）。留取肾脏组织制备病理切片,观察Gpx1-Klk1转基因小鼠抗肾缺血再灌注损伤的能力。 结果 获得全长为6.585 kb的pKsp-Gpx1-IRES-Klk1重组质粒,并证明了该克隆的正确性。在转基因小鼠制备过程中,共获得525枚注射卵,移植成功率为81.0%,小鼠出生总数109只。经基因组DNA的PCR检测,确认10只为转基因阳性小鼠,阳性率为9.2%。Western印迹法检测证实了阳性转基因小鼠肾脏组织有Gpx1和Klk1蛋白强表达。转基因小鼠（实验组）和野生型小鼠（对照组）肾缺血再灌注损伤模型建立后,实验组血样中尿素氮及肌酐水平显著低于对照组（P < 0.01）;肾脏组织中SOD显著高于对照组 （P < 0.01）,MDA显著低于对照组（P < 0.01）;两组肾组织中tNOS较缺血再灌注前均显著升高,且实验组显著高于对照组（P = 0.025）,实验组肾组织中iNOS显著低于对照组（P < 0.01）。缺血再灌注后,实验组肾组织间质轻度水肿,肾小管上皮坏死细胞较少,对标本损伤程度采用半定量评分后显示,实验组损伤程度显著轻于对照组（1.58±1.05比3.95±0.80,P < 0.05）。 结论成功制备Gpx1-Klk1转基因小鼠。Gpx1-Klk1过表达对肾缺血再灌注损伤的保护作用。     

成骨细胞特异性过表达h1 calponin转基因小鼠的建立

目的为从整体动物水平研究碱性调宁蛋白（h1-calponin）在骨形成过程中的直接作用，我们构建了成骨细胞中特异性过表达h1—calponin的转基因小鼠模型，并对其表型进行了初步分析。方法构建在成骨细胞特异性启动子（collagen I promoter）驱动下表达h1—calponin的载体（pColl-calponin），纯化DNA片段，再以显微注射的方法将转基因片段导人小鼠受精卵，经移植后得到小鼠。PCR法检测整合到小鼠基因组中的外源基因，提取F1代阳性小鼠原代成骨细胞RNA，RT-PCR检测h1-calponin在小鼠成骨细胞中的表达情况，并观察转基因小鼠表型及体重变化情况。结果PCR检测结果显示1只Go代转基因鼠中检测到阳性信号，RT-PCR显示F1代转基因小鼠成骨细胞中表达h1—calponin较野生对照小鼠明显增加，且转基因小鼠体重与野生小鼠相比有明显降低。结论成功构建了在成骨细胞特异性过表达h1—calponin的转基因小鼠，为深入研究h1—calponin在骨骼发育中的作用提供了良好的动物模型。     

可控性Speedy A基因表达转基因小鼠模型的建立鉴定和表型分析

目的建立基于Tet-On系统诱导表达的Speedy A基因RNA干扰转基因小鼠模型,探讨Tet-On系统在研究精子发生相关基因中的应用,研究Speedy A基因下调对精子发生的影响。方法体外实验筛选下调Speedy A基因表达的有效干扰片段,Gateway技术构建包含有效干扰片段和四环素反应元件(TRE)的质粒(pRP.EX2d-TREshSpdya),受精卵显微注射该质粒建立转基因小鼠并筛选到纯合子,然后与四环素调控的反式激活子(rtTA)转基因小鼠杂交,筛选TRE和rtTA双阳性的小鼠为四环素诱导的Speedy A-RNA干扰转基因小鼠模型,喂食强力霉素(Dox)诱导RNA干扰片段的表达下调Speedy A基因的表达;实时荧光定量PCR法检测模型组小鼠与喂食强力霉素的正常小鼠(对照组)Speedy A的表达差异;HE染色观察睾丸组织形态学变化,TUNEL法检测小鼠睾丸组织的细胞凋亡情况。结果模型组小鼠Speedy A基因的表达量与对照组小鼠的表达量相比下降26%;模型组小鼠睾丸组织发生异常的生精细胞凋亡。结论成功建立了基于Tet-On系统诱导表达的可控性Speedy A-RNA干扰转基因小鼠模型,说明可以基于Tet-On系统在体研究精子发生相关基因的功能;Speedy A基因表达下调可使生精细胞发生异常凋亡。     

Renal L-type fatty acid-binding protein mediates the bezafibrate reduction of cisplatin-induced acute kidney injury

Fibrates, the PPAR alpha ligand-like compounds increase the expression of proximal tubule liver fatty acid binding protein (L-FABP) and significantly decrease cisplatin-induced acute kidney injury. To study whether the bezafibrate-mediated upregulation of renal L-FABP was involved in this cytoprotective effect we treated transgenic mice of PPAR agonists inducible human L-FABP expression with cisplatin in the presence or absence of bezafibrate. Blood urea nitrogen was unchanged in the first day but increased 3 days after cisplatin. While urinary L-FABP increased over 100-fold 1 day after cisplatin treatment in the transgenic mice it was significantly reduced when these transgenic mice were pretreated with bezafibrate. Cisplatin-induced renal necrosis and apoptosis were significantly reduced in bezafibrate pretreated transgenic mice and this correlated with decreased accumulation of lipid and lipid peroxidation products. Immunohistochemical analysis of kidney tissue of bezafibrate-cisplatin-treated transgenic mice showed preservation of cytoplasmic L-FABP in the proximal tubule, but this was reduced in transgenic mice treated only with cisplatin. L-FABP mRNA and protein levels were significantly increased in bezafibrate-cisplatin-treated transgenic mice when compared to mice not fibrate treated. Our study shows that the bezafibrate-mediated upregulation of proximal tubule L-FABP plays a pivotal role in the reduction of cisplatin-induced acute kidney injury.     

Expression of the human renin gene in transgenic mice throughout ontogeny

Expression of a human renin genomic DNA clone extending 900 base pairs upstream and 400 base pairs downstream of the gene has been previously examined in adult transgenic mice. In adults, expression of human renin was evident in kidney, reproductive tissues, adrenal gland and lung. Previous studies of mouse and rat renin have demonstrated that kidney renin becomes evident at approximately 15 days of gestation and that expression is localized first to smooth muscle cells of the developing renal arterial tree and becomes progressively restricted to juxtaglomerular cells. As a prelude to performing cell specificity studies to elucidate the pattern of human renin gene expression in the developing kidney, 15.5 and 17.5 days of gestation fetuses and newborns were obtained for expression analysis. Tissues were pooled and expression was examined in kidney, liver, gastrointestinal (GI) tract, lung, heart and brain. The number of transgenic fetuses in each pool was determined by human renin-specific polymerase chain reaction of DNA purified from placenta or tail biopsies. Renal human renin expression was abundant at all three time points. Expression was also evident in the GI tract at 15.5 and 17.5 days of gestation. Interestingly, although no human renin mRNA was evident in lung at 15.5 or 17.5 days of gestation, extremely high levels of human renin mRNA were detected in the newborn lung. Expression of the human renin gene in these tissues was further confirmed by differential primer extension analysis which is capable of differentiating the closely related human and mouse renin messages. These transgenic mice should provide an interesting model to examine the expression and regulation of the human renin gene during kidney development.     

Targeting gene expression to endothelium in transgenic animals: a comparison of the human ICAM-2, PECAM-1 and endoglin promoters

Abstract: It is highly likely that successful pig-to-human xenotransplantation of vascularized organs will require genetic modification of the donor pig, and in particular of donor vascular endothelium. Promoters are generally tested in transgenic mice before generating transgenic pigs. Several promoters have been used to drive endothelial cell-specific expression in mice but none have yet been tested in pigs. We compared the promoters of three human genes that are predominantly expressed in vascular endothelium: intercellular adhesion molecule 2 (ICAM-2), platelet endothelial cell adhesion molecule 1 (PECAM-1) and endoglin. Expression of human complement regulatory proteins (hCRPs), directed by each of the promoters in mice, was largely restricted to vascular endothelium and leukocyte subpopulations. However, expression from the PECAM-1 promoter was weak in liver and non-uniform in the small vessels of heart, kidney, and lung. Conversely, expression from the endoglin promoter was consistently strong in the small vessels of these organs but was absent in larger vessels. The ICAM-2 promoter, which produced strong and uniform endothelial expression in all organs examined, was therefore used to generate hCRP transgenic pigs. Leukocytes from 57 pigs containing at least one intact transgene were tested for transgene expression by flow cytometry. Forty-seven of these transgenic pigs were further analyzed by immunohistochemical staining of liver biopsies, and 18 by staining of heart and kidney sections. Only two of the pigs showed expression, which appeared to be restricted to vascular endothelium in heart and kidney but was markedly weaker than in transgenic mice produced with the same batch of DNA. Thus, in this case, promoter performance in mice and pigs was not equivalent. The weak expression driven by the human ICAM-2 promoter in pigs relative to mice suggests the need for additional regulatory elements to achieve species-specific gene expression in pigs.     

转人CD46基因抑制人补体在转基因小鼠心脏组织中的沉积

目的 观察转人源性膜辅助蛋白(CD46)基因对转基因小鼠心脏中人补体沉积的抑制作用.方法 以近交系昆明小鼠为对象,采用显微注射法制备转人CD46基因小鼠,用逆转录聚合酶链法(RT-PCR法)检测外源基因的整合情况.以Fo代转基因小鼠11只为实验组,同窝非转基因小鼠10只为对照,快速切取小鼠心脏,用改良的Langendorff法经小鼠主动脉逆行灌注预先制备的含补体的B型血人血浆.观察并记录小鼠心脏搏动时间;采用免疫荧光和免疫组织化学法检测小鼠心脏组织中补体C3c及C9的沉积情况.结果 Fo代转基因小鼠外源基因的整合率为33.3%.实验组小鼠心脏搏动时间为(42.6±20.6)min(15～77 min),长于对照组的(20.2±12.5)min(7～40 min),差异有统计学意义(P＜0.01).实验组小鼠心脏组织中补体C3c及C9的沉积均少于对照组.结论 通过显微注射法制备的转人CD46基因小鼠,其外源基因可稳定表达;转人CD46基因可以抑制人补体C3c及C9在转基因小鼠心脏组织中的沉积.     

Skeletal abnormalities and ultrastructural changes of cartilage in transgenic mice expressing a collagen II gene (COL2A1) with a Cys for Arg-alpha1-519 substitution

OBJECTIVE: To examine the mechanism by which the Arg-->Cys 519 mutation causes the clinical phenotype employing transgenic mice that express the mutated human COL2A1. METHODS: A DNA construct under the control of a COL2A1 specific promoter was prepared from genomic DNA isolated from fibroblasts from the proband with primary generalized osteoarthritis (OA) associated with a mild chondrodysplasia. Transgenic mice were obtained by injection of the constructs into pro-nuclei of fertilized eggs from the FVB/N inbred mouse strain. Transgenic mice harboring two alleles of the mutated human COL2A1 were examined for morphological abnormalities and for alterations of their skeletal development. Ultrastructural examination was performed to identify changes in the organization and density of collagen II fibrils in articular cartilage of the transgenic mice. RESULTS: Transgenic mice harboring two alleles of the mutated human collagen gene were smaller than their normal littermates, had a cleft palate, and disorganized growth plate. Electron microscopy of articular cartilage showed a decreased density of collagen II fibrils and revealed chondrocytes with dilated Golgi cysternae. CONCLUSIONS: Expression of a COL2A1 with an Arg-->Cys 519 substitution in transgenic mice causes retardation of skeletal development and ultrastructural alterations in articular cartilage with a profound reduction of the density of the collagen II fibrils in the tissue. These alterations may be responsible for the phenotype of precocious generalized OA and chondrodysplasia displayed by patients harboring this COL2A1 mutation.     

Glomerulosclerosis in mice transgenic for human insulin-like growth factor-binding protein-1

BACKGROUND: The growth hormone (GH)/insulin-like growth factor (IGF) system is thought to participate in the glomerulosclerosis process. Because IGF-binding proteins (IGFBPs) modulate IGF actions and hence GH secretion, this study assessed whether mice transgenic for human IGFBP-1 have altered susceptibility to glomerulosclerosis. METHODS: A line of transgenic mice that express human IGFBP-1 mRNA in the liver under the control of the alpha1-antitrypsin promoter has been obtained, and morphological changes in the kidney tissue were assessed. Glomerulosclerosis was identified using light microscopy, light microscopic morphometry, and electron microscopy. Extracellular matrix components were analyzed by immunohistochemistry. RESULTS: There was a marked increase in mesangial extracellular matrix area in homozygous transgenic mice at three months of age as compared with heterozygous transgenic mice and nontransgenic littermates. These changes were not associated with alterations in glomerular volume or cellularity. The expansion of extracellular matrix area was related to a marked increase in laminin and type IV collagen and to the appearance of type I collagen. CONCLUSIONS: These observations indicate that the enhanced expression of IGFBP-1 may result in the development of glomerulosclerosis without glomerular hypertrophy. The changes are potentially related to a decrease in IGF-I availability and/or to an IGF-I-independent role of IGFBP-1.     

Human CLC-KB gene promoter drives the EGFP expression in the specific distal nephron segments and inner ear

Human CLC-KB has been identified as a kidney-specific member of the CLC chloride channel family, and mutations of the human CLC-KB gene are known to cause Bartter syndrome type III. A precise understanding of the localization of this channel in the human kidney is imperative to our understanding of the pathophysiology, but this has remained unclear due to the high homology between human CLC-KB and CLC-KA, another kidney-specific member of the same family. The high intraspecies homology also rules out an exact correlation of the human isoforms (CLC-KA and CLC-KB) to the mouse and rat isoforms (CLC-K1 and CLC-K2, respectively). This study created transgenic mice harboring the enhanced green fluorescence protein (EGFP) gene driven by an 11-kbp human CLC-KB gene promoter. Three transgenic lines were generated, and all of them showed EGFP fluorescence in the kidney, with an identical pattern of localization to the thick ascending limb of Henle's loop, distal tubules, connecting tubules, and intercalated cells of the collecting duct. This localization is exactly the same as that of mouse CLC-K2 identified in a previous report (Kobayashi et al. J Am Soc Neph 12: 1327-1334, 2001). EGFP fluorescence was also detected in the inner ear, more specifically in marginal cells of the stria vascularis and dark cells of the vestibular labyrinth, suggesting that human CLC-KB could play an important role in the fluid transport mechanism of the inner ear. The results (1) confirmed that CLC-KB is the true human homologue of rat and mouse CLC-K2 and (2) established that the 11-kbp human CLC-KB gene promoter is sufficient to elicit the precise expression in specific cell types of the kidney and inner ear.