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
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
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DOI：
10.1002/cbin.10942
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发表时间：
2018-05-01
影响因子：
3.9
通讯作者：
Tomita, Hiroshi
中科院分区：
文献类型：
--
作者：
Fukuda, Tomokazu;Ishizawa, Yasushi;Tomita, Hiroshi
四种关键转录因子（CRX、RAX、NEURO-D、OTX 2）的引入允许成纤维细胞直接分化为视网膜感光细胞。这种重编程是用单顺反子病毒的组合实现的。虽然单顺反子病毒的组合是有用的，但由于需要共感染，因此需要相对高滴度的重组病毒。为了克服这个问题，我们建立了一个多顺反子表达系统，直接重编程，并分析了外源导入后导入细胞的生物学特性。将四种重编程因子和EGFP（CRX、RAX、NEURO-D、OTX 2和EGFP; CNROE）的编码区插入pMYs-IP逆转录病毒或CSII-CMV慢病毒载体的多个位点。将重组病毒暴露于HE 16人胚胎成纤维细胞。实时荧光定量PCR检测视锥细胞相关基因的表达水平。在CRX、RAX、NEURO-D和OTX 2的多顺反子表达后，我们检测到两个光感受器相关基因的激活，但其余基因没有表现出转录升高。我们得出结论，CNROE的多顺反子表达诱导部分重编程进入感光细胞。我们推测，直接重编程到感光细胞可能需要相对较高的蛋白质表达水平的转录因子。
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.