May 2006
Volume 47, Issue 13
ARVO Annual Meeting Abstract  |   May 2006
Characterization of Pre–RNA Processing Factor 3 (Prpf3) Knockin Mice
Author Affiliations & Notes
  • J.J. Graziotto
    University of Pennsylvania, Philadelphia, PA
  • C.F. Inglehearn
    Molecular Medicine Unit, University of Leeds, Leeds, United Kingdom
  • E.A. Pierce
    University of Pennsylvania, Philadelphia, PA
  • Footnotes
    Commercial Relationships  J.J. Graziotto, None; C.F. Inglehearn, None; E.A. Pierce, None.
  • Footnotes
    Support  Zeigler Foundation, FFB, RPB, F.M. Kirby Foundation, Mackall Foundation Trust, Wellcome Trust Grant 073988
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 4588. doi:
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      J.J. Graziotto, C.F. Inglehearn, E.A. Pierce; Characterization of Pre–RNA Processing Factor 3 (Prpf3) Knockin Mice . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4588.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Retinitis Pigmentosa (RP) 18 is one of 4 forms of RP caused by mutations in pre–RNA splicing factors, which as a group constitute the second leading cause of dominant RP. Missense mutations (T494M;P493S) in PRPF3 cause RP18 through an unknown mechanism. It is not known how mutations in the ubiquitous PRPF3 can cause retina specific disease. We have generated Prpf3 knockin mice to study the disease mechanism of RP18.

Methods: : We isolated a 12Kb fragment of Prpf3 from a mouse genomic library using homologous recombination. We performed site–directed mutagenesis to insert the T494M mutation, added a selection cassette for ES cell targeting and electroporated ES cells with this knockin targeting vector. We screened resistant ES cells via Southern blotting, followed by sequencing, and injected a positive ES cell clone into blastocysts to generate chimeric founder mice, from which F1 and F2 generations were derived. The amount of Prpf3 in the retina and brain was assessed by Northern and Western blotting. Visual function was evaluated by electroretinograph (ERG) analysis. The retina was examined using light, immunofluorescent, and electron microscopy. The expression and splicing of mRNAs for RP disease genes was evaluated by Northern Blotting.

Results: : We confirmed correct homologous recombination at the Prpf3 locus by Southern blot analysis and sequencing. Germline transmission of the knockin (KI) allele was achieved from 2 of 8 chimeric founders tested. To date, all hetero– and homozygous KI mice are healthy and fertile. Expression of the mutant transcript was confirmed by RT–PCR followed by sequencing. Western blot analysis revealed that the level of mutant Prpf3 protein was normal. ERG analyses of wildtype (WT), WT/KI and KI/KI mice at 5 months and WT/KI vs. WT/WT mice at 1 year reveal no alterations in visual function. No changes in retinal histology have been identified to date. Northern blotting revealed no differences in the splicing of rhodopsin mRNA in KI/KI vs. WT/WT mice.

Conclusions: : The Prpf3 T494M KI allele in both hetero– and homozygous states appears not to affect the retina of mice at timepoints of up to one year. Continued evaluation of these mice at additional time points is in progress. Additionally, it appears that rhodopsin mRNA is spliced correctly in the retinas of KI mice. The splicing of other RP disease genes, and mRNA splicing in general, are currently under investigation in the knockin mice.

Keywords: retinal degenerations: hereditary • retinal degenerations: cell biology • transgenics/knock-outs 

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