Apical gene transfer into quiescent human and canine polarized intestinal epithelial cells by lentivirus vectors

Abstract

Intestinal epithelial cells secrete a protective luminal mucus barrier inhibiting viral gene transfer. Quiescent, polarized monolayers of primary epithelial cells from dog gallbladder and human colon are efficiently transduced through the apical mucus side by lentivirus vectors, suggesting their application to intestinal gene therapy.

FIG. 1

(A) BrdU labeling of DGBE cells. Confluent DGBE cells were labeled for 24 h with BrdU, and incorporation was detected using a mouse monoclonal anti-BrdU antibody and tetramethyl rhodamine isothiocyanate-conjugated anti-mouse immunoglobulins. Nuclei that have incorporated BrdU can be identified by their red fluorescence. A representative field is shown. Magnification, ×10. (B) Frozen cross sections of transwell membranes with transduced DGBE cells. Transwell membranes with DGBE cells transduced by pRtatpeGFP were fixed in 4% paraformaldehyde, embedded in Tissue-Tek O.C.T. compound (Sakura Finetek, Torrance, Calif.), frozen for 1 h at −80°C, and sectioned in a cryostat. A monolayer of cells covering the transwell membrane can be observed in a representative field with simultaneous fluorescent and bright-field illumination. Magnification, ×10.

FIG. 2

Transduction of DGBE and CaCo-2 cells with lentivirus and murine retroviral vectors. DGBE and CaCo-2 cells were transduced and processed for microscopy. Exposure times were 10 s for all fields except DGBE cells transduced with pRtatpeGFP, which were exposed for 5s. The top two rows represent CaCo-2 cells, and the bottom two rows represent DGBE cells. The virus used and the polarity of infection are indicated above the panels. AZT, transduction with first-generation lentivirus from the apical compartment and simultaneous treatment with reverse transcriptase inhibitor AZT. DAPI, representative field of DAPI staining showing nuclei of all cells present. Magnification, ×10.