Article Figures & Data
Figures
- 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.
Tables
- Table 1.
Transduction of dividing CaCo-2 and DGBE cellsa
Cells and vector % GFP-positive cells Mean fluorescence intensity CaCo-2 Untransfected 2.5 23.1 pLNCeGFP 44 140.6 pRtatpeGFP 99 2,781 pRRLcmveGFPsin 76 287.6 DGBE Untransfected 1 22.6 pLNCeGFP 89 2,229 pRtatpeGFP 87 2,498 pRRLcmveGFPsin 96 1,609 a Subconfluent CaCo-2 and DGBE cells were infected with murine retrovirus LNCeGFP, first-generation lentivirus pRtatpeGFP, and third-generation lentivirus pRRLcmveGFPsin. Cells were analyzed by flow cytometry. All viral vectors transduced target cells efficiently. Mean fluorescence levels are similar in DGBE cells, and pRtatpeGFP mediates higher expression levels in CaCo-2 cells.
- Table 2.
Transduction of DGBE and CaCo-2 cells with lentivirus and murine retroviral vectorsa
Cells and vector % GFP-positive cellsb Apical infection Basolateral infection CaCo-2 pLNCeGFP 2.6 ± 2 <0.5 pRtatpeGFP 43 ± 17 4.7 ± 1 pRRLcmveGFPsin 24 ± 16 <0.5 DGBE pLNCeGFP 2.9 ± 1 2.4 ± 1 pRtatpeGFP 33 ± 25 <0.5 pRRLcmveGFPsin 3.8 ± 0.5 <0.5 a Confluent monolayers of DGBE and CaCo-2 cells were transduced with first- and third-generation lentivirus vectors and a murine retroviral vector. Experiments were performed using at least two different viral preparations per virus type.
b Results are averages and standard deviations from at least three different independent experiments.
