Gene transfer to human trabecular meshwork cells in vitro and ex vivo using HIV-based lentivirus

Xiang, Yan, Li, Bin, Wang, Jun-Ming, Li, Gui-Gang, Zhang, Hong, Manyande, Anne and Tian, Xuebi (2014) Gene transfer to human trabecular meshwork cells in vitro and ex vivo using HIV-based lentivirus. International Journal of Ophthalmology, 7 (6). pp. 924-929. ISSN 2222-3959

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Abstract

AIM: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 1×108 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).

CONCLUSION:EGFP gene could be efficiently transferred into HTM cells both in vitro and ex vivo by using HIV-based lentivirus.

Item Type: Article
Additional Information: IJO Press applies the Creative Commons Attribution (CC BY NC ND) license to the works we publish including IJO and IES. This license was developed to facilitate open access.
Uncontrolled Keywords: gene transfer, trabecular meshwork, HIV-based lentivirus, glaucoma
Subjects: Medicine and health > Clinical medicine > HIV/AIDS epidemiology
Medicine and health > Clinical medicine
Medicine and health
Depositing User: Anne Manyande
Date Deposited: 09 May 2017 13:45
Last Modified: 23 Jun 2017 10:41
URI: http://repository.uwl.ac.uk/id/eprint/3315

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