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Federica Polato, Luigi Notari, Lijin Dong, Patricia Becerra; A conditional knockout mouse for Pnpla2 in photoreceptors. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4633.
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© ARVO (1962-2015); The Authors (2016-present)
Pigment epithelium-derived factor (PEDF), an extracellular multifunctional protein with neurotrophic properties, exerts its activity by binding to cell-surface receptors. We have identified PEDF-R, a protein encoded by the PNPLA2 (Patatin-Like Phospholipase Domain Containing Protein 2) gene as a PEDF receptor critical for the prosurvival activity of PEDF that is located in photoreceptors. The homologous PEDF binding domain in mouse PEDF-R is encoded by exon 4. The biological role of PEDF-R in vivo has not been studied yet. The purpose of this study is to generate and characterize a conditional Pnpla2 knockout mouse model on photoreceptors.
Homozygous Pnpla2 floxed (Pnpla2f/f) mutant mice, carrying LoxP sites flanking exons 2-4 of the Pnpla2 gene, were generated by homologous recombination in ES cells and backcrossed for 5 generations to C57Bl/6 background. Genomic and genotyping PCR were performed using specifically designed primers. The Crx-Cre transgenic mouse line was used to create homozygous for the floxed allele also carrying the Crx-Cre allele (Pnpla2f/f/Cre). PEDF-R was detected by immunoblotting of total protein extracts prepared from whole retinas. Pnpla2 transcripts levels were determined in retinas dissected from 1 eye per mouse by semi-quantitative PCR (qPCR), using primers homologous to either the floxed region or downstream exons 2-4. Electroretinogram (ERG) analysis was performed.
Homologous recombination in ES cells to introduce LoxP sites was positively confirmed by genomic PCR. Pnpla2f/f mice were crossed with Crx-Cre transgenic mouse line in which the expression of the Cre gene is under the control of the promoter of the cone-rod homeobox (Crx) gene, active in developing photoreceptors and pinealocytes. Both Pnpla2f/f and Pnpla2f/f/Cre mice were viable, normal in size, fertile with no visible physical or behavioral abnormalities. Pnpla2 transcripts and PEDF-R levels in whole retinas from Pnpla2f/f/Cre and control littermates showed similar variations. However, functional evaluation of retinas by ERG analysis carried out on knockout (n=8) and control littermates revealed that in Pnpla2f/f/Cre mice the amplitude of the scotopic a- and b-wave as well as the photopic b-wave was attenuated relative to controls.
Although genomic mosaic after Crx-Cre-mediated excision might be occurring, Pnpla2-null photoreceptors partially impaired the functionality of the retina of mice in vivo.
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