Cloning of the Rat Ecotropic Retroviral Receptor and Studies of Its Expression in Intestinal Tissues

Abstract

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.