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T. Wang, F. A. Concepcion, H. Moaven, J. Chen; Restoring CNGB Expression With Cre Recombinase in a CNGB-/-Mouse Line. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4039.
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© ARVO (1962-2015); The Authors (2016-present)
Retinitis pigmentosa (RP) is a form of retinal degeneration that affects nearly 1 in 3000 people globally. For all retinal rescue strategies, retinal remodeling as a consequence of degeneration constitutes a major challenge, and the potential of functional recovery after such remodeling events is an important question to address. The CNGB-/- mice is a good RP model exhibiting similar retinal degeneration patterns as human RP. Here, we have generated a CNGB-/- mouse line that is capable of CNGB gene reactivation upon Cre-mediated recombination. These mice will allow for a systematic study of the extent of vision recovery following defined periods of retinal degeneration.
The Loxp-flanked neomycin resistance (LBL) cassette was introduced into the endogenous CNGB1 locus by homologous recombination. The targeting vector was engineered to introduce a mutation within the CNGB1 gene that deletes its calmodulin binding site. Importantly, the insertion of the targeting vector precludes CNGB gene expression, and expression of CNGB1ΔCaM can be achieved by excision of the LBL cassette by the Cre recombinase. Mice that expressed CNGB1ΔCaM were created following germline excision of the LBL cassette. Visual function was assessed by electroretinogram. The retinal structures at different ages were investigated by light and electron microscopy as well as by immunocytochemistry.
The removal of LBL cassette led to the expression of the CNGB1ΔCaM protein. The CNGB1ΔCaM protein expressed at the same level as WT CNGB1, and is located at the rod outer segment plasma membrane. The dark-adapted CNGB1ΔCaM mice showed normal flash sensitivities and light adaption, while the CNGB-/- mice had little or no measurable rod response. Similar to WT, the CNGB1ΔCaM mice maintained normal retinal structure up to 12 month. In contrast, retinas from CNGB1-/- mice show a moderate rate of degeneration which was complete by 3 months.
Our data showed that the regulated activation of the CNGB1ΔCaM protein is feasible, and the CNGB1ΔCaM protein can form a functional CNG channel at the rod outer segment and restore normal light responses. Therefore, the CNGB-/- mice can be conditionally activated to express the functional channel. This will allow for the systematic study of the extent of functional recovery following different stages of retinal degeneration. The conditional activation of CNGB1ΔCaM will be achieved by crossing the CNGB-/- mice with transgenic mice that express a ligand-inducible ERT2CreERT2 construct in the rod photoreceptors
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