Type II pneumocyte-restricted green fluorescent protein expression after lentiviral transduction of lung epithelial cells.
Type II alveolar epithelial (AT2) cell-specific reporter expression has been highly useful in the study of embryology and alveolar regeneration in transgenic mice. Technologies enabling efficient gene transfer and cell type-restricted transgene expression in AT2 cells would allow for correction of AT2 cell-based diseases such as genetic surfactant deficiencies. Moreover, such approaches are urgently required to investigate differentiation of AT2 cells from adult and embryonic stem cells of other species than mouse. Using a human surfactant protein C (SP-C) promoter fragment, we have constructed lentiviral vectors enabling AT2-restricted transgene expression and identification of stem cell-derived AT2 cells. Lung epithelial cell lines M3E3/C3, H441, RLE-6TN, A549, MLE-12, and MLE-15 were characterized at the molecular and ultrastructural levels to identify cell lines useful to assess the cell type specificity of our vector constructs. After transduction, no green fluorescent protein (GFP) expression was observed in nontarget cells including bronchial H441 cells, pulmonary A549 cells, fibroblasts, smooth muscle cells, and endothelial cells. In contrast, and in correlation with endogenous SP-C expression, lentiviral transduction resulted in stable GFP expression in MLE-12 and MLE-15 AT2 cells. In conclusion, we have constructed a lentiviral vector mediating SP-C promoter-dependent GFP expression. Transgene expression strictly corresponds with an AT2 phenotype of the transduced cells. In particular, the generated vector should facilitate local alveolar gene therapy and investigation of alveolar regeneration and stem cell differentiation.
Leibniz Research Laboratories for Biotechnology and Artificial Organs, Hannover Medical School, 30659 Hannover, Germany.
Stephanie
S
Wunderlich
Ina
I
Gruh
Monica E
ME
Winkler
Jennifer
J
Beier
Kerstin
K
Radtke
Andreas
A
Schmiedl
Stephanie
S
Groos
Axel
A
Haverich
Ulrich
U
Martin
D000818:Animals; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000378:metabolism; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D010641:Phenotype; D011401:Promoter Regions, Genetic; D011650:Pulmonary Alveoli / Q000166:cytology* / Q000378:metabolism*; D037721:Pulmonary Surfactant-Associated Protein C / Q000235:genetics*; D051381:Rats; D014161:Transduction, Genetic; D019076:Transgenes
D037721:Pulmonary Surfactant-Associated Protein C; D049452:Green Fluorescent Proteins