Rakoczy P E, Lai M C, Baines M G, Spilsbury K, Constable I J
Department of Molecular Biology, Centre for Ophthalmology and Visual Science, University of Western Australia, Nedlands, Australia.
Invest Ophthalmol Vis Sci. 1998 Oct;39(11):2095-104.
To show the production of sense or antisense transcripts by recombinant adenoviruses, to investigate whether the transcripts produced were suitable for downregulating the expression of the targeted gene, cathepsin S (CatS), and to examine the effect of antisense transcript production on the biologic function of retinal pigment epithelial (RPE) cells, including the regulation of endogenous aspartic protease expression.
Ad.MLP.CatSAS, Ad.RSV.CatSAS, and Ad.MLP.CatSS recombinant viruses were produced by homologous recombination. The recombinant viruses were tested by restriction enzyme digestion to confirm the orientation of the inserts. The expression of antisense transcripts was tested by northern blot analysis. Western blot analysis was used to study the regulation of the endogenous CatS protein in ARPE19 cells. The biologic effect of CatS downregulation in ARPE19 cells was tested by proliferation and phagocytosis assays, de novo cathepsin D (CatD) synthesis, and measurement of aspartic protease activity.
After characterization of the recombinant adenovirus constructs, the production of antisense and sense CatS transcripts was shown in ARPE19 cells. The transcripts appeared at approximately 1.9 kb 48 hours after transduction, and the expression of the antisense transcripts was similar in constructs carrying either the MLP or the RSV promoter. Western blot analysis showed that ARPE19 cells transduced with Ad.MLP.CatSAS and Ad.RSV.CatSAS had no detectable CatS. In contrast, there was a strong signal appearing at 24 kDa in ARPE19 cells transduced with Ad.MLP.CatSS. ARPE19 cells were transduced to a high level. The transduction of ARPE19 cells with the recombinant adenoviruses did not affect the morphologic appearance of the cells, their proliferation, or their phagocytosing ability. However, ARPE19 cells transduced by Ad.MLP.CatSAS recombinant adenovirus showed a significant downregulation of de novo CatD synthesis and a twofold decrease in aspartic protease activity.
Recombinant adenoviruses were shown to be suitable for producing antisense CatS transcripts to modulate endogenous CatS expression in RPE cells. It is proposed that CatS may play an important role, directly or indirectly, in the lysosomal digestion of outer segments through the regulation of other lysosomal enzyme activity, such as the expression of CatD.
展示重组腺病毒产生的正义或反义转录本，研究产生的转录本是否适合下调靶基因组织蛋白酶S（CatS）的表达，并检测反义转录本的产生对视网膜色素上皮（RPE）细胞生物学功能的影响，包括对内源性天冬氨酸蛋白酶表达的调控。
通过同源重组产生Ad.MLP.CatSAS、Ad.RSV.CatSAS和Ad.MLP.CatSS重组病毒。通过限制性酶切对重组病毒进行检测，以确认插入片段的方向。通过Northern印迹分析检测反义转录本的表达。采用蛋白质印迹法研究ARPE19细胞中内源性CatS蛋白的调控情况。通过增殖和吞噬试验、组织蛋白酶D（CatD）的从头合成以及天冬氨酸蛋白酶活性测定，检测ARPE19细胞中CatS下调的生物学效应。
对重组腺病毒构建体进行鉴定后，在ARPE19细胞中显示出反义及正义CatS转录本的产生。转导后48小时，转录本出现在约1.9 kb处，并且在携带MLP或RSV启动子的构建体中，反义转录本的表达相似。蛋白质印迹分析显示，用Ad.MLP.CatSAS和Ad.RSV.CatSAS转导的ARPE19细胞中未检测到CatS。相反，用Ad.MLP.CatSS转导的ARPE19细胞在24 kDa处出现强信号。ARPE19细胞被高效转导。用重组腺病毒转导ARPE19细胞不影响细胞的形态外观、增殖或吞噬能力。然而，用Ad.MLP.CatSAS重组腺病毒转导的ARPE19细胞显示出从头合成CatD的显著下调以及天冬氨酸蛋白酶活性降低两倍。
已证明重组腺病毒适合产生反义CatS转录本，以调节RPE细胞中的内源性CatS表达。有人提出，CatS可能通过调节其他溶酶体酶活性（如CatD的表达），直接或间接在外段的溶酶体消化中起重要作用。
