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Z. Xinrong, Y. Huiping, S. Zhengbo; Construction and Package of Recombinant Retrovirus PLXSN-BDNF and RSC Labelled by GFP Subretinal Space Transplantion. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6098.
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© ARVO (1962-2015); The Authors (2016-present)
Objective: To construct the recombinant retroviral expression vector pLXSN-BDNF carrying BDNF gene. Screened a stable cell line secreting recombinant virus for the protection of optic nerve injury through neural stem cells transplantation. To observe the cells growth and immigration after Rat Schwann cells (RSCs) labelled by EGFP transplanted into subretinal space.
BDNF gene was amplified by RT-PCR. It was cloned into the retrovirus vector pLXSN. The genome DNA was detected by PCR and the recombinant virus were evaluated by RT-PCR. The titer of retrovirus was determined by fluorescent quantitative RT-PCR and clone formation method after it was concentrated. RSCs transfected by pEGFP-N1 which could express GFP, then transplanted into SD subretinal space after 24 h. Observed the growth and implantation of RSCs at different time after transplantation by HRA,OCT and cryocut.
BDNF gene was correctly cloned. With PCR, restriction analysis and DNA sequencing, the BDNF was inserted into the vector exactly. The plasmid pLXSN-BDNF was transfected into the package cell line and G418-resistant clones were selected. The recombinant virus pLXSN-BDNF was secreted into the cell supernatant. The titer of recombinant retrovirus was 6.92×106 copies/ml by fluorescent quantitative PCR; while it was 3.2×105 CFU/ml by clone formation method after it was concentrated. The cell was observed by HRA,OCT and cryocut after RSCs labelled by EGFP were transplanted into the rat subretinal space. The transplanted cells were seen indistinctly at region at 1day after operation. The cells became more at 3 day. After 2 weeks, single cells could be seen. After 4 weeks, not only at transplanted area, but also at the near district, cells can be found. We resumed that the transplanted RSCs maybe growed well in vivo, and had a near immigration. The cells still could be seen after 6 weeks, but there was only a little.
The BDNF gene had been amplified successfully, and the recombinant palsmid pLXSN-BDNF was constructed correctly. The stable cell line producing the recombinant retrovirus carrying BDNF gene had been established. A novel quantitative PCR assay had been developed to replace the standard clone formation method for retroviral titer. The transplanted RSCs were well in the region, and had a near migration. The recombinant retrovirus pLXSN-BDNF and transplantation of RSCs may lay foundations for a further study of gene therapy experiment for curing the optic nerve injury due to retinal diseases such as glaucoma.
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