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Chen Lin, Dustin Whitaker, Hirva Bakeri, Chunqiao Liu, Anand Swaroop, Tiansen Li; Functional Characterization Of Prickle1 Gene In Mouse Retina. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4922.
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© ARVO (1962-2015); The Authors (2016-present)
Prickle1 is a core component of the planar cell polarity (PCP) pathway and regulates multiple processes during development. However, its function in mammalian retina is unknown. Complete ablation of prickle1 gene in mice causes early embryonic lethality. To further investigate Prickle1 function in retina, we studied Prickle1 expression in developing and adult tissues and created several Prickle1 mouse mutants that are viable, allowing detailed functional analysis of the mutant retina.
Hypomorphic gene-trap, conditional, and straight knockout alleles were generated. A gene-trap mutant allele was created by introduction of an eYFP reporter with En1 splicing acceptor 5’ of the 2nd exon of Prickle1 gene before the initiation codon. Three loxP and two Frt sites were arranged such that a complete Cre excision will lead to deletion of exon 2 to generate a straight knockout allele, while Flp excision will remove the eYFP reporter retaining a loxP-flanked exon 2 to generate a conditional knockout allele. Prickle1 protein expression in retinal neurons was monitored by the eYFP reporter, and the residual levels of Prickle1 in different mutants were determined by immunoblotting. IHC was used to localize expression of Calretinin, Islet1, Brn3a, NFL and Sox9. Electroretinograms (ERGs) were recorded to examine changes in retinal function.
Immunoblot analysis detected different levels of Prickle1 expression in various mutant alleles. eYFP reporter showed Prickle1 is expressed in amacrine, bipolar, and ganglion cells. The eYFP reporter expression is consistent with Prickle1 mRNA expression detected by in situ hybridization. Mice homozygous for Prickle1 gene-trap allele show normal ERG a and b waves at one month of age, while mice carrying one straight knockout and one excised conditional allele die within a week of birth. Further investigations of retinal functions using mice carrying different combinations of mutant alleles are in progress.
We generated several mutant alleles of Prickle1, offering opportunities to study the role of Prickle1 in retinal development and function over a range of gene dosages. Using the eYFP reporter, we defined Prickle1-expressing neurons in the retina. Further analyses of retinal and systemic phenotypes of the mutant(s) are in progress.
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