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.

Authors:
Tomokazu Fukuda
Tomokazu Fukuda
Tohoku University
Japan
Yasushi Ishizawa
Yasushi Ishizawa
Graduate School of Science and Engineering
Kenichiro Donai
Kenichiro Donai
Tohoku University
Japan
Eriko Sugano
Eriko Sugano
Tohoku University
Japan
Hiroshi Tomita
Hiroshi Tomita
Nihon University School of Medicine
Japan

Cell Biol Int 2018 May 20;42(5):608-614. Epub 2018 Feb 20.

Graduate School of Science and Engineering, Iwate University, 4-3-5, Ueda, Iwate Prefecture, 020-8551, Morioka, Japan.

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.

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http://dx.doi.org/10.1002/cbin.10942DOI Listing
May 2018
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