title: Gene transfer to human trabecular meshwork cells in vitro and ex vivo using HIV-based lentivirus.
abstract: OBJECTIVE
  To investigate whether the enhanced green fluorescent protein (EGFP) reporter gene could be transferred into human trabecular meshwork (HTM) cells by a HIV-based lentivirus both in vitro and ex vivo.
  
  
  METHODS
  The HIV-based lentivirus that contains an EF1-α promoter driving EGFP expression cassette was constructed following the standard molecular cloning methods. The cultured HTM cells were transduced at a range of multiplicity of infection (MOI) with HIV-based lentivirus. EGFP positive cell populations were detected by flow cytometry. Human anterior eye segments were cultured with perfusion culture system and transfected by HIV-based lentivirus with a 1×10(8) transducing unit (TU) virus in perfusion liquid. The intraocular pressure was recorded every 8h for 21d. The expression of EGFP in the anterior segment of the human eye was detected by fluorescence microscopy. Furthermore, the distribution of EGFP expression was confirmed by anti-EGFP immunohistochemical staining.
  
  
  RESULTS
  The HIV-based lentivirus which contains an EF1-α promoter driving EGFP expression cassette was constructed successfully. After HTM cells were transduced with HIV-based lentivirus containing EGFP in vitro, the ratio of EGFP positive cells to the total cell number reached 92.3%, with the MOI of 15. After the lentivirus containing EGFP were used to transduce human anterior eye segments, the EGFP could be directly detected by fluorescence microscopy in vivo. Immunohistochemistry staining revealed that 88.19% EGFP-positive trabecular meshwork (TM) cells were observed in the human anterior segment. Nevertheless, the intraocular pressure in the lentivirus-transduced group kept constant when compared with control group (P>0.05).
  
  
  CONCLUSIONS
  EGFP gene could be efficiently transferred into HTM cells both in vitro and ex vivo by using HIV-based lentivirus.
authors.affiliation: Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Xiang
authors.affiliation: Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Li
authors.affiliation: Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China.
authors.forename: Jun-Ming
authors.initials: JM
authors.lastname: Wang
authors.affiliation: Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China.
authors.forename: Gui-Gang
authors.initials: GG
authors.lastname: Li
authors.affiliation: Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Zhang
authors.affiliation: School of Psychology, Social Work and Human Sciences, University of West London, London W5 5RF, UK.
authors.forename: Anne
authors.initials: A
authors.lastname: Manyande
authors.affiliation: Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China.
authors.forename: Xue-Bi
authors.initials: XB
authors.lastname: Tian
keywords: HIV-based lentivirus; gene transfer; glaucoma; trabecular meshwork