April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
Germline Transmission and Retinal Expression of Human P23H Rhodopsin in a Miniature Swine Model of Retinitis Pigmentosa (RP)
Author Affiliations & Notes
  • J. W. Ross
    Animal Science, Iowa State University, Ames, Iowa
  • R. S. Prather
    Animal Science,
    University of Missouri, Columbia, Missouri
  • E. Walters
    Veterinary Pathobiology,
    University of Missouri, Columbia, Missouri
  • C. Rios
    Pediatrics, Genetics Laboratory, University of Nevada School of Medicine, Las Vegas, Nevada
  • P. Bray-Ward
    Pediatrics, Genetics Laboratory, University of Nevada School of Medicine, Las Vegas, Nevada
  • H. J. Kaplan
    Ophthalmology and Visual Sciences, University of Louisville, Louisville, Kentucky
  • Footnotes
    Commercial Relationships  J.W. Ross, None; R.S. Prather, None; E. Walters, None; C. Rios, None; P. Bray-Ward, None; H.J. Kaplan, None.
  • Footnotes
    Support  NIH U42-RR18877, Discovery Eye Foundation, Research to Prevent Blindness
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 2944. doi:
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      J. W. Ross, R. S. Prather, E. Walters, C. Rios, P. Bray-Ward, H. J. Kaplan; Germline Transmission and Retinal Expression of Human P23H Rhodopsin in a Miniature Swine Model of Retinitis Pigmentosa (RP). Invest. Ophthalmol. Vis. Sci. 2010;51(13):2944.

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Abstract

Purpose: : The objective of this investigation was to determine the germline transmission and retinal expression of a human P23H rhodopsin (RHO) transgene in the progeny of a founder transgenic mini swine from our newly established inbred (SLAcc haplotype) colony.

Methods: : Fluorescent in situ hybridization (FISH) of chromosome spreads was used to identify the number of integration sites for each of the founder mini swine that were positive by PCR analysis. Sperm from one transgenic founder (53-1), demonstrating early onset of RP, was used to inseminate a domestic wild-type pig and a NIH cc wild-type pig. Progeny were genotyped via PCR for the presence of the human RHO transgene. Nine progeny resulted from the insemination of the domestic pig and six from the NIH mini swine. The offspring from the domestic pig were sacrificed at one day of age. Total RNA from their retinae was utilized for cDNA synthesis, PCR amplification and DNA sequencing to determine the ratio of human vs. pig RHO transcript expression in the retina.

Results: : FISH analysis showed a single integration site on different chromosomes for each founder. For the two litters produced by 53-1, five of nine piglets from the domestic sow and 2 of 6 piglets from the mini pig sow were positive for the transgene. Transgenic offspring expressed 4-8 copies of the human P23H RHO transcript for each endogenously expressed pig RHO transcript.

Conclusions: : These data suggested that the human RHO transgene integrated in different genomic regions in the founder transgenic minipigs. The transgene was transmitted to the progeny of 53-1 in Mendelian fashion and expressed in their retinae more abundantly than the endogenous porcine RHO mRNA. Germline transmission of the P23H rhodopsin mutation in this inbred NIH mini swine model provides us with a manageably-sized large animal model of human RP.

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