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Cell Biol Int. 2018 May;42(5):608-614. doi: 10.1002/cbin.10942. Epub 2018 Feb 20.

The poly-cistronic expression of four transcriptional factors (CRX, RAX, NEURO-D, OTX2) in fibroblasts via retro- or lentivirus causes partial reprogramming into photoreceptor cells.

Author information

1
Graduate School of Science and Engineering, Iwate University, 4-3-5, Ueda, Iwate Prefecture, 020-8551, Morioka, Japan.
2
Soft-Path Engineering Research Center (SPERC), Iwate University, Morioka, Japan.
3
Graduate School of Agricultural Science, Tohoku University, Sendai, Japan.

Abstract

The introduction of four key transcriptional factors (CRX, RAX, NEURO-D, OTX2) allows the direct differentiation of fibroblasts to retinal photoreceptor cells. This reprogramming was achieved with a combination of mono-cistronic viruses. Although the combination of mono-cistronic viruses was useful, a relatively high titer of recombinant viruses was necessary because co-infections are required. To overcome this issue, we established a poly-cistronic expression system for direct reprogramming and analyzed the biological characteristics of introduced cells after the exogenous introduction. The coding region of four reprogramming factors and EGFP (CRX, RAX, NEURO-D, OTX2, and EGFP; CNROE) was inserted into multiple sites of the pMYs-IP retrovirus or CSII-CMV lentivirus vector. The recombinant viruses were exposed to HE16 human embryonic fibroblasts. The expression levels of cone related genes were detected with real-time PCR. We detected the activation of two of the photoreceptor-related genes after the poly-cistronic expression of CRX, RAX, NEURO-D, and OTX2, but the rest of the genes did not exhibit transcriptional elevation. We concluded that the poly-cistronic expression of CNROE induced partial reprogramming into photoreceptor cells. We hypothesize that the direct reprogramming into photoreceptor cells might require relatively high protein expression levels of transcriptional factors.

KEYWORDS:

direct reprogramming; photoreceptor cells; poly-cistronic expression; recombinant virus; transcriptional factor

PMID:
29377330
DOI:
10.1002/cbin.10942
[Indexed for MEDLINE]

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