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GENERATION OF STABLE Caco-2/15 CELL LINES DEPLETED OF INTEGRIN alpha7Bbeta1 USING shRNA TECHNOLOGY
A Seltana, N Basora, JF Beaulieu
Equipe IRSC sur l’epithelium digestif Departement d’anatomie et biologie cellulaire, Universite de Sherbrooke, Sherbrooke, Quebec
INTRODUCTION: Cell matrix interactions play a critical role in the control of intestinal epithelial cell differentiation. We have previously shown that the presence of laminin-10 in the basal membrane regulates cell differentiation of Caco-2/15 cells, and these cells become polarized, form extensive cell-cell junctions and show up-regulation of the expression of intestinal epithelial cell markers, such as sucrase-isomaltase. We have also shown that the distribution of one particular laminin-binding integrin isoform, alpha7Bbeta1, is directly correlated to the initiation of cell differentiation in vivo. In the foetal small and large intestine as well as in the adult small intestine, alpha7Bbeta1 is found to be located at the crypt-villus junction, where committed cells initiate their differentiation program.
OBJECTIVE: To determine the role of the alpha7Bbeta1 integrin in intestinal epithelial cell differentiation.
RESULTS: We used the Caco-2/15 cell line as an in vitro model of cell differentiation. These cells express only the alpha7B isoform, as determined by RT-PCR and western analyses. Interestingly, the expression of alpha7B increases as cells reach confluency and levels decrease as differentiation progresses. In order to determine the impact of alpha7B in intestinal cell differentiation we generated a stable Caco-2/15 cell line depleted of the alpha7B chain using shRNA technology.
Five different shRNA sequences were tested in Caco-2/15 cells by lentiviral infection and selected for 10 days using puromycin. Western analyses of whole cell lysates showed that the different shRNAs were successful in depleting the integrin alpha7B chain to various degrees. The most successful cell lines were obtained by using a mixture of two different shRNA sequences. Western analyses using a specific anti-alpha7B antibody, directed against the cytoplasmic domain, showed significant depletion of the 120kD immature protein (50%) and complete knock down of the mature form.
CONCLUSION: Expression of the alpha7Bbeta1 integrin correlates with the onset of epithelial cell differentiation. The generation of a cell model depleted for this integrin will allow us to directly determine the role of alpha7Bbeta1 in proliferation and differentiation signalling pathways.