Functional analisis of invgene which determines lef-right axis and relates to renal development

脊椎动物的内脏器官通常是左右不对称的。而胸腹各不配对的器官，如心、胃、脾、肝等，在胎儿时均从中线发育，在成人时定位到正常位置。这种不对称的镜像反转称为位置反转。通过随机插入诱变，在小鼠体内产生了胚胎逆转（tnv）突变。inv小鼠的表型是左-右极性（倒置位置）和肾脏囊性形成的一致镜像反转。在之前的项目中，我们成功克隆了小鼠inv的全序列。由于候选基因编码的蛋白涉及锚蛋白基序的15个连续重复，因此可能存在细胞骨架异常参与肾脏结构异常、倒置和囊肿形成的机制和产生。基于测序数据，我们对inv基因有了新的认识，主要有：1)成功克隆了人类inv基因，确定了基因组结构和染色体定位。2)用GFP蛋白或lacZ等报告基因修饰小鼠inv基因，并插入表达载体中。本研究拟采用全鼠基因表达载体在培养细胞中进行转染分析。3)用GFP蛋白或lacZ等部分报告基因修饰小鼠inv基因，排除外显子1 ~ 2，构建敲除小鼠的靶载体。

Vertebrate organisms have a common left-right asymmetry of their visceral organs. However, all unpaired organs of the chest and abdomen, such as heart, stomach, spleen and liver, develop from the midline in the fetus and localize to their normal positions in the adult. The mirror image reversal of this asymmetry is called situs inversus. The inversion of embryonic turning (tnv) mutation in a mouse was created by random insertional mutagenesis. The phenotype of the inv mouse is a consistent mirror-image reversal of the left-right polarity (situs inversus) and cystic formation of the kidneys. In previous project, we succeeded in cloning the whole sequence of mouse inv. As protein encoded by a candidate gene involved 15 consecutive repeats of ankyrin motif, there may be a possibility that a cytoskeletal abnormality was involved in the mechanism and production of structural abnormalities, inversion and cyst formation in the kidney. Based on the sequencing data, we had new results as to inv gene summarized below :1) We succeeded in cloning the human inv gene and determined genomic organization, chromosomal mapping.2) We modified mouse inv gene with some reporter genes, such as GFP protein or lacZ, and inserted into the expression vectors. We intend to use expression vector mounted whole mouse inv gene for transfection analysis in the culture cells.3) We modified mouse inv gene with some reporter genes, such as GFP protein or lacZ, and excluded exon 1 to 2 for made up targeting vector for knockout mouse.