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The newt (Cynops pyrrhogaster) RPE65 promoter: molecular cloning, characterization and functional analysis.

ABSTRACT: The adult newt has the ability to regenerate the neural retina following injury, a process achieved primarily by the retinal pigment epithelium (RPE). To deliver exogenous genes to the RPE for genetic manipulation of regenerative events, we isolated the newt RPE65 promoter region by genome walking. First, we cloned the 2.8 kb RPE65 promoter from the newt, Cynops pyrrhogaster. Sequence analysis revealed several conserved regulatory elements described previously in mouse and human RPE65 promoters. Second, having previously established an I-SceI-mediated transgenic protocol for the newt, we used it here to examine the -657 bp proximal promoter of RPE65. The promoter assay used with F0 transgenic newts confirmed transgene expression of mCherry fluorescent protein in the RPE. Using bioinformatic tools and the TRANSFAC database, we identified a 340 bp CpG island located between -635 and -296 bp in the promoter; this region contains response elements for the microphthalmia-associated transcription factor known as MITF (CACGTG, CATGTG), and E-boxes (CANNTG). Sex-determining region box 9 (or SOX9) response element previously reported in the regulation of RPE genes (including RPE65) was also identified in the newt RPE65 promoter. Third, we identified DNA motif boxes in the newt RPE65 promoter that are conserved among other vertebrates. The newt RPE65 promoter is an invaluable tool for site-specific delivery of exogenous genes or genetic manipulation systems for the study of retinal regeneration in this animal.

SUBMITTER: Casco-Robles MM

PROVIDER: S-EPMC4436847 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Retinal regeneration in the adult newt is a useful system to uncover essential mechanisms underlying the regeneration of body parts of this animal as well as to find clues to treat retinal disorders such as proliferative vitreoretinopathy. Here, to facilitate the study of early processes of retinal regeneration, we provide a de novo assembly transcriptome and inferred proteome of the Japanese fire bellied newt (Cynops pyrrhogaster), which was obtained from eyeball samples of day 0-14 after surgical removal of the lens and neural retina. This transcriptome (237,120 in silico transcripts) contains most information of cDNAs/ESTs which has been reported in newts (C. pyrrhogaster, Pleurodeles waltl and Notophthalmus viridescence) thus far. On the other hand, de novo assembly transcriptomes reported lately for N. viridescence only covered 16-31% of this transcriptome, suggesting that most constituents of this transcriptome are specific to the regenerating eye tissues of C. pyrrhogaster. A total of 87,102 in silico transcripts of this transcriptome were functionally annotated. Coding sequence prediction in combination with functional annotation revealed that 76,968 in silico transcripts encode protein/peptides recorded in public databases so far, whereas 17,316 might be unique. qPCR and Sanger sequencing demonstrated that this transcriptome contains much information pertaining to genes that are regulated in association with cell reprogramming, cell-cycle re-entry/proliferation, and tissue patterning in an early phase of retinal regeneration. This data also provides important insight for further investigations addressing cellular mechanisms and molecular networks underlying retinal regeneration as well as differences between retinal regeneration and disorders. This transcriptome can be applied to ensuing comprehensive gene screening steps, providing candidate genes, regardless of whether annotated or unique, to uncover essential mechanisms underlying early processes of retinal regeneration.

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The newt (Cynops pyrrhogaster) RPE65 promoter: molecular cloning, characterization and functional analysis.

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