Independent degeneration of photoreceptors and retinal pigment epithelium in conditional knockout mouse models of choroideremia
Tanya Tolmachova, … , Clare Huxley, Miguel C. Seabra
Tanya Tolmachova, … , Clare Huxley, Miguel C. Seabra
Published February 1, 2006
Citation Information: J Clin Invest. 2006;116(2):386-394. https://doi.org/10.1172/JCI26617.
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Independent degeneration of photoreceptors and retinal pigment epithelium in conditional knockout mouse models of choroideremia

Abstract

Choroideremia (CHM) is an X-linked degeneration of the retinal pigment epithelium (RPE), photoreceptors, and choroid, caused by loss of function of the CHM/REP1 gene. REP1 is involved in lipid modification (prenylation) of Rab GTPases, key regulators of intracellular vesicular transport and organelle dynamics. To study the pathogenesis of CHM and to develop a model for assessing gene therapy, we have created a conditional mouse knockout of the Chm gene. Heterozygous-null females exhibit characteristic hallmarks of CHM: progressive degeneration of the photoreceptors, patchy depigmentation of the RPE, and Rab prenylation defects. Using tamoxifen-inducible and tissue-specific Cre expression in combination with floxed Chm alleles, we show that CHM pathogenesis involves independently triggered degeneration of photoreceptors and the RPE, associated with different subsets of defective Rabs.

Authors

Tanya Tolmachova, Ross Anders, Magnus Abrink, Laurence Bugeon, Margaret J. Dallman, Clare E. Futter, José S. Ramalho, Felix Tonagel, Naoyuki Tanimoto, Mathias W. Seeliger, Clare Huxley, Miguel C. Seabra

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Figure 1

Generation of mice carrying the conditional and KO Chm alleles.

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Generation of mice carrying the conditional and KO Chm alleles. ...
(A) Targeting vector pTT55 carrying 3 loxP sites, a neomycin and spectinomycin resistance cassette (Neor, Spr), and 2 homology arms was used to generate the Chm3lox allele in GSI-1 ES cells by homologous recombination. Diagnostic HindIII and SstI restriction sites and corresponding 3′ and 5′ probes were used to identify correctly targeted ES clones. Cre-mediated recombination between the 3 loxP sites within the Chm3loxallele resulted in 3 possible alleles: Chmflox, Chmnull+Neo, and Chmnull, which were distinguished by Southern blot analysis using EcoRI digestion and probe A. (B) Results of Southern blot analysis using HindIII digestion and the 3′ probe, and SstI digestion and the 5′ probe, are shown for 4 correctly targeted ES clones (78, 92, 147, and 197) and a wild-type ES clone. (C) Results of Southern blot analysis using EcoRI digestion and probe A. The Chm alleles of the mice are indicated above the lanes. “MCM” indicates mice carrying the MerCreMer transgene. “+TM” indicates treatment with TM.