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Run-Tao Yan, Wenjie zhan, Li He, Shu-Zhen Wang; Constructing a lenti vector to drive neurogenin3 expression in RPE cells for mammalian retinal regeneration. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3645.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal regeneration in the mammalian eye remains an elusive goal. Our laboratory studies an unconventional approach of reprogramming the RPE for photoreceptor regeneration in the eye. Previous studies have shown the presence of ectopic retinal tissue/cells in some of the transgenic mice generated with DNA constructs expressing proneural gene neurogenin1 (ngn1) or ngn3 from an RPE promoter (PVMD2 or PRPE65). This study aims to generate a gene delivery vehicle compatible with future human application of our scheme for retinal regeneration in the eye.
A 3-step engineering was carried out upon a commercial lentiviral vector pLvx-ZsGreen1-C1: (i) removing the PPGK-Puror to make space for PVMD2-ngn3; (ii) inserting PVMD2 at the multiple-cloning-site of the vector; and (iii) inserting the sequence of human ngn3 downstream of the PVMD2. Both the DNA construct and viral particles were tested for biological activities with primary RPE cell cultures. The culture was monitored through epi-fluorescence imaging of ZsGreen1 (ZG) over days post transfection or infection to observe transition of RPE cells into cells with neuronal morphologies.
The new construct, named pLvx-ZG-PVMD2-ngn3-s, offers the opportunity to confine ngn3 expression to PVMD2-active cells (to initiate photoreceptor differentiation program in the cells) and to turn off ngn3 once reprogramming takes place (to facilitate the maturation of the new cells into functional photoreceptors). It enables the tracking of the cells through epi-fluorescence of ZG (driven by PCMV). DNA transfection and/or viral infection of primary RPE cell cultures derived from embryonic chick, adult mouse, and juvenile pig resulted in de novo generation of cells displaying morphologies typical of neurons. Some of the neuron-like, ZG+ cells contained pigment granules, reminiscent of cells in a transitional stage of RPE-to-neuron reprogramming.
A novel expression vector, pLvx-ZG-PVMD2-ngn3-s, has been generated. Being vector-based and with a tracer (ZG), the new vector makes it feasible to study how the RPE gives rise to retinal neurons and regenerates itself, information critically important to potential clinical applications of our reprogramming scheme.
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