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X. Fan, S. Hao, X. Liu, M. L. Robinson, V. M. Monnier; An Animal Model to Study the Dynamic Regulation of Glutathione in the Lens: The Lenticular Gamma Glutamate-Cysteine Ligase Conditional Knockout Mouse (Preliminary Studies). Invest. Ophthalmol. Vis. Sci. 2010;51(13):4769.
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Glutathione (GSH) is the most abundant small molecular thiol compound found in aerobic eukaryotic organism. It fulfils numerous cellular functions, including protection against oxidative stress, control of cellular redox potential and detoxification of xenobiotics. GSH is also a principle antioxidant keeping lens crystallin damage in check. Lens is maintaining very high level of GSH reaching millimolar concentrations. Biochemical synthesis of GSH occurs through the sequential action of two ATP-dependent enzymes: γ-glutamate cysteine ligase (Gcl) and glutathione synthase (GS). The systematic knockout of Gcl leads to embryonic lethality in mammals and is not useful for lenticular studies.
The Cre-Lox site-specific recombination system was used by flanking γ-glutamate cysteine ligase catalytic subunit (Gclc) exon 2 with two Loxp sites developed by European Conditional Mouse Mutagenesis Program. The Flp-Frt site-specific recombination was used to flank Neo cassette, and eventually was removed via cross-breeding with Flp transgenic mouse. LoxP floxed Gclc mice was cross-bred with MRL-10 lens Cre transgenic mouse to create the lens conditional Gclc knockout mouse. The breeding was selected by regular PCR with specific primers through tail genomic DNA extraction. The Gclc gene expression and protein level were determined by real time PCR and Western-blot respectively.
Heterozygous knockout mouse showed 30-45% drop in mRNA level of Gclc and about 40-50% drop of protein level in lens compared to wild type mice. Surprisingly, the GSH level only dropped around 10% compared to wild type lenses. These results indicated that the dynamic regulation of GSH in lens occurs both by de novo synthesis and extracellular uptake via GSH transporter which has been reported before. The compensation from GSH transport indicates presence of a high affinity transporter in lens epithelial cells, because the GSH concentration in aqueous is in micromolarl, while in lens in millimolar level.
A conditional ko mouse for GSH synthesis in the lens has been created as an initial step for the study of impaired GSH levels in nuclear sclerosis. The heterozygous mouse shows only a modest drop in GSH that is compensated by lenticular uptake via a yet unknown high affinity transporter.
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