Biological function microRNAs enriched in RPE: in vitro and in vivo models

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MicroRNA (miRNA) is a group of short (1925 nucleotide), non-coding RNAs that are capable of downregulate gene expression by base pairing with the 3 untranslated regions (3UTRs) of target mRNAs. The structure of miRNAs is highly conserved across many species, indicating important function of these molecules. We have identified several miRNAs that express in relative high levels in human fetal retinal pigment epithelium (hfRPE) compared to retina and choroids. These potential regulatory enriching miRNAs miR-184, miR-187, miR-200, miR-221/222, miR-204, miR-211 -- were found to be associated with the maintenance of total tissue resistance of the RPE, suggesting the importance of these miRNAs in RPE barrier function. Despite their relative enrichment, detailed functions of microRNAs in RPE remain to be determined. Developing microRNA knockout mice will facilitate the understanding invivo function of individual microRNA. As an initial approach, we produced gene-targeting constructs that are designated to ablate individual microRNA constitutively from mouse. The gene segment that encodes individual microRNA was replaced with a neomycin resistant gene expression cassette via homologous recombination. Since a pair of the Lox-p sites flanks neo gene, the neo gene that is bearing within targeted allele can be eliminated by Cre-Lox-p mechanism. We can encounter following possible scenarios: As individual miRNAs can be specifically linked to critical developmental pathways, we will examine if knocking down a particular microRNA will cause any structure abnormalities in RPE and other tissues where the microRNA has shown expression. If function of a microRNA is pivotal to development, embryonic lethality can happen, then we will produce conditional knockout mice where a microRNA will be eliminated from RPE. If function of a miRNA is to fine-tune gene expression "noise" elicited by the environmental stress. We will determine the relevant stimulations and corresponding disorders. If a microRNA is resided within intron of a gene, its expression and function are often associated with its host gene. Therefore, the function of a microRNA will be studied in association with its host gene. In any case, RPE from knockout mice will be cultured and used as invitro model system to test loss of function and corresponding rescue strategies.