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Avigail Beryozkin, Alexey Obolensky, Segev Meyer, Ayala Ejzenberg, Chen Matsevich, Yvan Arsenijevic, Carlo Rivolta, Eyal Banin, Dror Sharon; Generation and characterization of Fam161a Conditional Knockout mice. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3151.
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© ARVO (1962-2015); The Authors (2016-present)
FAM161A mutations are the most common cause of inherited retinal degenerations in Israel. In addition, FAM161A is an excellent candidate for gene therapy: the open reading frame is relatively short and all reported mutations are null. A previous established Fam161a KO model showed retinal degeneration but a week expression level of the truncated protein in the retina. Our main purpose is to generate a line of knockout mice in which there is no Fam161a expression and examine the effect on retinal function and structure.
Mice with a heterozygous conditional non-activated construct were produced and used to generate a knock out line of mice that are homozygous for the activated allele by Cre activation. Mice were examined at different ages (1,3 and 6 months). The retinal localization of the construct was evaluated by LacZ staining. Retinal function was evaluated by electroretinography (ERG) in scotopic and photopic conditions and retinal structure was studied by optical coherence tomography (OCT) and histological analysis.
As part of the process of generating the KO line, we identified a known CRB1 mutation in the breeding colony and bred the mice with C57BL/6J mice in order to filter out this mutation. Subsequently, the mice were inbred with Cre mice aiming to create a line with an activated construct resulting in the deletion of the major Fam161a exon (exon #3). LacZ staining revealed that Fam161a is expressed in the ganglion cells, inner and outer nuclear layer in these mice. In photoreceptors, LacZ expression was noted in the inner and outer segments. ERG analysis revealed decrease in a- and b-wave amplitudes comparing to WT mice at ages 1, 3, and 6 months (n=8 animals per group). Light-adapted ERG showed lower responses that are correlated with light intensity. OCT analysis of the same tested animals at ages 3 and 6 months showed a minor difference between KO and WT animals in outer nuclear layer (ONL) thickness while major differences were observed at the age of 6 months. H&E histological analysis revealed a progressive degeneration of photoreceptors along time.
The results indicate that a homozygous Fam161a frameshift mutation affects retinal function and cause retinal degeneration. This model will be used for gene therapy treatment in the future.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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