Jiang J X, Goodenough D A
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760, USA.
Invest Ophthalmol Vis Sci. 1998 Mar;39(3):537-43.
To develop an in vivo model system in which exogenous proteins can be expressed in embryonic chick lens and to further understand the function of connexin-mediated gap junction intercellular communication in lens cell biology.
RCAS(A) is a replication-competent chicken retrovirus that infects dividing cells. Retroviral constructs were prepared containing alkaline phosphatase (AP) and FLAG-tagged connexins. Chick lenses were infected in situ by injecting virus into the lumen of lens vesicles at stage 18, cultures were taken at various periods. The lenses were then dissected, and the expressed proteins were visualized by AP histochemical examination and immunostaining.
Twenty-four hours after infection, alkaline phosphatase could be seen in epithelia and fibers. As lens fiber maturation progressed, however, the alkaline phosphatase staining was lost as the fibers matured, presumably because of the proteolytic removal of the enzyme. By 72 hours, alkaline phosphatase staining could still be observed in epithelial cells and in differentiating fibers in the bow region but not in the mature lens fibers. FLAG-tagged exogenous lens connexins were also abundantly expressed by viral infection. The exogenous connexins were localized at the cell surfaces in junctional maculae and showed the same cell-type specific distribution as that of their endogenous connexin counterparts.
An in vivo model system has been developed in the chick that provides opportunities to study the expression of wild-type and mutant proteins during lens differentiation. Expression of wild-type connexins has revealed that the characteristic distribution of the three different lens connexins is maintained even when expression is driven by a viral promoter.
建立一种体内模型系统，使外源蛋白能在胚胎鸡晶状体中表达，并进一步了解连接蛋白介导的缝隙连接细胞间通讯在晶状体细胞生物学中的功能。
RCAS(A)是一种具有复制能力的鸡逆转录病毒，可感染分裂细胞。制备了含有碱性磷酸酶(AP)和FLAG标签连接蛋白的逆转录病毒构建体。在第18阶段通过向晶状体泡腔内注射病毒对鸡晶状体进行原位感染，在不同时期取材培养。然后解剖晶状体，通过AP组织化学检查和免疫染色观察表达的蛋白。
感染后24小时，在上皮细胞和纤维细胞中可见碱性磷酸酶。然而，随着晶状体纤维成熟，碱性磷酸酶染色随着纤维成熟而消失，推测是由于该酶被蛋白水解去除。到72小时时，仍可在上皮细胞和弓状区域正在分化的纤维细胞中观察到碱性磷酸酶染色，但在成熟的晶状体纤维中未观察到。通过病毒感染也大量表达了FLAG标签的外源晶状体连接蛋白。外源连接蛋白定位于连接斑的细胞表面，显示出与其内源性连接蛋白对应物相同的细胞类型特异性分布。
已在鸡中建立了一种体内模型系统，该系统为研究晶状体分化过程中野生型和突变蛋白的表达提供了机会。野生型连接蛋白的表达表明，即使由病毒启动子驱动表达，三种不同晶状体连接蛋白的特征性分布仍得以维持。
