title: Development and limitations of lentivirus vectors as tools for tracking differentiation in prostate epithelial cells.
abstract: To investigate hierarchy in human prostate epithelial cells, we generated recombinant lentiviruses, infected primary cultures and cell lines, and followed their fate in vitro. The lentiviruses combined constitutive promoters including CMV and β-actin, or late-stage differentiation promoters including PSCA (prostate stem cell antigen) and PSAPb (prostate specific antigen/probasin) driving expression of monomeric, dimeric and tetrameric fluorescent proteins. Significantly, rare CD133(+) cells from primary prostate epithelial cultures were successfully infected and activation of late-stage promoters was observed in basal epithelial cultures following induction of differentiation. Lentiviruses also infected CD133(+) cells within the P4E6 cell line. However, promoter silencing was observed in several cell lines (P4E6, BPH-1, PC3). We examined the promoter methylation status of the lentiviral insertions in heterogeneously fluorescent cultures from PC3 clones and found that DNA methylation was not the primary mechanism of silencing of the CMV promoter. We also describe limitations to the lentivirus system including technical challenges due to low titers and low infection efficiency in primary cultures. However, we have identified a functional late-stage promoter that indicates differentiation from a basal to a luminal phenotype and demonstrate that this strategy for lineage tracking of prostate epithelial cells is valid with further optimisation.
authors: YCR Cancer Research Unit, Department of Biology, University of York, Heslington, North Yorkshire, YO10 5DD, UK. ff502@york.ac.uk
authors: Fiona M
authors: FM
authors: Frame
authors: Stefanie
authors: S
authors: Hager
authors: Davide
authors: D
authors: Pellacani
authors: Mike J
authors: MJ
authors: Stower
authors: Hannah F
authors: HF
authors: Walker
authors: Julie E
authors: JE
authors: Burns
authors: Anne T
authors: AT
authors: Collins
authors: Norman J
authors: NJ
authors: Maitland
mesh_terms: D000071916:AC133 Antigen; D015703:Antigens, CD / Q000378:metabolism; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D002999:Clone Cells; D004847:Epithelial Cells / Q000166:cytology* / Q000821:virology; D005453:Fluorescence; D020868:Gene Silencing; D017930:Genes, Reporter; D005821:Genetic Techniques; D005822:Genetic Vectors / Q000235:genetics*; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D016180:Lentivirus Infections / Q000821:virology; D008297:Male; D010455:Peptides / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D011467:Prostate / Q000166:cytology*; D017430:Prostate-Specific Antigen / Q000235:genetics; D011471:Prostatic Neoplasms / Q000473:pathology / Q000821:virology; D017422:Sequence Analysis, DNA
chemical_list: D000071916:AC133 Antigen; D015703:Antigens, CD; D006023:Glycoproteins; C000606023:PROM1 protein, human; D010455:Peptides; D017430:Prostate-Specific Antigen