Analysis of the lymphatic development using mutant mice

淋巴管被认为是导致患者水肿症的原因，尤其是患有原发性淋巴水肿症的患者。最近的研究表明，淋巴管生成和血管生成在肿瘤转移中也起着一定的作用。然而，由于缺乏可靠的解剖学/组织学方法来检测淋巴管，以及具有明显淋巴管异常的有用突变动物，哺乳动物的淋巴管发育一直未知。为了了解淋巴管发育和功能的机制，我们通过转基因或靶向突变的方法，利用淋巴管内皮细胞特异性基因转录调控下的标记基因(LacZ、GFP、PLAP)，如LYVE-1、PROX-1和VFGFR-3，来产生报告菌株。我们克隆了小鼠LYVE-1基因，并测定了该基因在小鼠体内的基因组结构和染色体定位。为了构建使用Prox-1基因或VEGFR-3基因的转基因，我们购买了它们的P1或BAC克隆并进行了重新分析。我们现在正在向小鼠受精卵中引入一些结构。我们还开始了淋巴管生成中潜在的生长因子/受体和转录因子的条件性基因敲除分析。我们还在研究一种原始的自发突变小鼠系，它们会出现乳糜性腹水、肠道淋巴水肿和水肿性后足肿胀，这些都被认为是淋巴异常所致。在纯合子突变体中，不仅在血管中发现了血流，而且在淋巴管中也发现了血流。根据TRITC结合明胶注入血管的共聚焦显微镜分析，肠绒毛处的外周毛细血管-乳房分流是纯合子突变小鼠淋巴管血流观察的原因之一。我们正在尝试先进的遗传学方法来寻找这种突变的候选基因(S)。

The lymphatics are thought to be responsible for edematous condition in patients, especially in patients suffering from primary lymphedema. Resent studies show that lymphangiogenesis, as well as angiogenesis, also plays some roles on tumor metastasis. However, the lymphatic development in mammals has been unknown from lack of reliable anatomical / histological procedure for the detection of lymphatic vassals and useful mutant animal that has obvious lymphatic abnormality.In order to understand the mechanism of lymphatic development and functions, we are generating "reporter" strains by transgenesis or targeted mutagenesis, using marker genes (lacZ, GFP, PLAP) under transcriptional regulation of the lymphatic endothelial-specific genes, such as LYVE-1, prox-1 and VFGFR-3. We have cloned mouse LYVE-1 gene and determined the genomic structure and chromosomal location of the gene in mice. For the construction of the transgenes using Prox-1 gene or VEGFR-3 gene, we purchased and re-analyzed their P1 or BAC clones. We are now introducing some constructs into mouse fertilized eggs. We have also started on the conditional knockout analyses of potential growth factor / receptor and transcriptional factors in lymphangiogenesis.We are also under investigation of an original spontaneous mutant mouse line developing chylous ascites, intestinal lymphedema and edematous hindfoot that are thought to be due to lymphatic abnormality. The blood flow is found not only in blood vessels but also in lymphatic vessels in the homozygous mutants. According to the confocal microscopic analysis of TRITC-conjigated gelatin injected into blood vessels, the peripheral capillary-lacteal shunt at the intestinal villi is one of the cause for blood flow observed lymphatics of the homozygous mutant mice. We are trying forward genetic approaches to find the candidate gene (s) for this mutation.