title: Feline immunodeficiency virus-mediated long-term transgene expression in undifferentiated retinal progenitor cells and its downregulation in differentiated cells.
abstract: OBJECTIVE
  Lentivirus-mediated gene transfer is an important approach to modify the function of progenitor cells in ex vivo gene therapy, but may be susceptible to downregulation due to transcriptional silencing. The purpose of this study was to analyze the stability of lentivirus-mediated transgene expression in undifferentiated and differentiated retinal progenitor cells (RPCs), and to characterize the effect of lentivirus transduction on RPC differentiation in vitro.
  
  
  METHODS
  RPCs derived from postnatal day 1 mice were expanded in defined serum-free culture medium and transduced with nonprimate lentiviral vector of feline immunodeficiency virus (FIV) expressing yellow fluorescent protein (YFP) reporter. Long-term expression of YFP in undifferentiated and differentiated RPCs was analyzed. Expression of various markers for RPCs and differentiated cells was analyzed by immunochemical staining in lentivirus-transduced and control RPCs. Differentiated postmitotic cells were revealed by negative labeling of bromodeoxyuridine (BrdU).
  
  
  RESULTS
  FIV transduction induced long-term expression of YFP reporter in RPCs for up to 53 days (10 passages) with no sign of decrease in expression level. FIV transduction did not alter the expression profile of various markers in retinal spheres, including nestin, microtubule-associated protein 2 (MAP-2), glial fibrillary acidic protein (GFAP), and opsin. However, YFP expression was downregulated in differentiated BrdU-negative postmitotic cells.
  
  
  CONCLUSIONS
  FIV-mediated long-term expression of transgene in undifferentiated RPCs is downregulated upon their differentiation. Thus, lentivirus-mediated ex vivo modulation should be cautiously analyzed for transgene expression not only in undifferentiated RPCs, but also in differentiated postmitotic cells.
authors.affiliation: Bascom Palmer Eye Institute, Department of Ophthalmology, University of Miami School of Medicine, Miami, FL 33136, USA.
authors.forename: Branislava
authors.initials: B
authors.lastname: Janic
authors.forename: Xuxiang
authors.initials: X
authors.lastname: Zhang
authors.forename: Wei
authors.initials: W
authors.lastname: Li
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002415:Cats; D002454:Cell Differentiation / Q000502:physiology; D015536:Down-Regulation; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D016089:Immunodeficiency Virus, Feline / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008938:Mitosis; D012160:Retina / Q000166:cytology*; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism*; D013997:Time Factors; D019076:Transgenes
chemical_list: D008164:Luminescent Proteins