Puppi M, Henning S J
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030-3498, USA.
Proc Soc Exp Biol Med. 1995 May;209(1):38-45. doi: 10.3181/00379727-209-43875.
A long-term goal of our laboratory is to establish a rat model to study the feasibility of using the intestinal tract as a site for somatic gene therapy. As a step toward that goal, the current study reports the cloning of the rat ecotropic retroviral receptor (EcoR) cDNA and the study of various aspects of its expression in the intestinal tissues. The cDNA for rat EcoR was cloned by screening a size-selected rat intestinal cDNA library with mouse EcoR cDNA. A clone of approximately 7 kb, designated MP10, was obtained. Partial sequencing of MP10 from the 5' end revealed a level of similarity of 92% compared with mouse EcoR. The presence of a 5' untranslated region and a 3' poly(A) tract, together with the overall size of the cDNA, suggest that is very close to being a full-length cDNA for this large transcript. Northern blots with MP10 showed an RNA of approximately 7.9 kb present along the entire length of the small intestine and somewhat less abundant in the colon. Developmental studies showed high levels of EcoR in fetal rat intestine, a decline in the early postnatal period, then a gradual rise to adulthood. Caco-2 cells were used to assess the expression of EcoR in proliferating compared with differentiated intestinal epithelial cells. EcoR mRNA was found to be very much more abundant in nondifferentiated cells and declined to low levels as the cells underwent spontaneous differentiation. These patterns of EcoR expression indicate that ecotropic retroviruses should be suitable vectors with which to attempt gene transfer into the intestinal epithelium. In addition, since the endogenous role of EcoR is as the y+ cationic amino acid transporter, these data have significance for understanding patterns of amino acid transport in the intestinal epithelium.
我们实验室的一个长期目标是建立一种大鼠模型，以研究将肠道作为体细胞基因治疗位点的可行性。作为朝着该目标迈出的一步，当前研究报告了大鼠嗜亲性逆转录病毒受体（EcoR）cDNA的克隆及其在肠道组织中表达的各个方面的研究。通过用小鼠EcoR cDNA筛选大小选择的大鼠肠道cDNA文库，克隆了大鼠EcoR的cDNA。获得了一个约7 kb的克隆，命名为MP10。从5'端对MP10进行部分测序，发现与小鼠EcoR的相似性水平为92%。5'非翻译区和3'聚腺苷酸尾的存在，以及cDNA的整体大小，表明它非常接近这个大转录本的全长cDNA。用MP10进行的Northern印迹显示，在小肠全长均存在约7.9 kb的RNA，在结肠中的丰度略低。发育研究表明，大鼠胎儿肠道中EcoR水平较高，出生后早期下降，然后逐渐上升至成年期。使用Caco-2细胞评估增殖的与分化的肠上皮细胞中EcoR的表达。发现EcoR mRNA在未分化细胞中丰富得多，随着细胞自发分化而下降至低水平。这些EcoR表达模式表明，嗜亲性逆转录病毒应该是适合尝试将基因转移到肠上皮的载体。此外，由于EcoR的内源性作用是作为y+阳离子氨基酸转运体，这些数据对于理解肠上皮中氨基酸转运模式具有重要意义。
