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Eri Kubo, Teppei Shibata, Shinsuke Shibata, Hiroshi Sasaki, Etsuko Kiyokawa, Masato Ikawa, Dhirendra P Singh; Knock-down of Tropomyosin 2 created by CRISPR-CAS9 in mouse induces age-related-cataract. Invest. Ophthalmol. Vis. Sci. 2016;57(12):No Pagination Specified. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The tropomyosin (Tpm) family of cytoskeleton proteins is involved in regulating and stabilizing actin microfilaments (F-actin). Previously we demonstrated that elevated expression of Tpm1α/2β was related to progression of rat posterior capsule opacification (PCO) after cataract surgery. In this study, we investigated the role of Tpm2 in lens by generating Tpm2 hetero-knock-out (Tpm2+/-) mice, using the clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) system.
Experiments accorded with National Institutes of Health Guide for the Care and Use of Laboratory Animals. We generated CRISPR/CAS9 -mediated Tpm2 mutations in germline C57BL/6NCr mice. In mice aged 7-, 16- and 26-weeks old, lenses were extracted (n=6eyes), and observed by microscope. The expression levels of mRNA and protein were examined by western blot and real-time RT PCR, respectively. Tissues in paraffin sections were histologically analyzed.
Tpm2+/- mice were selected because the Tpm2 gene null-knockout mouse exhibits viviparous lethality. At 7- and 16-weeks old, lenses were transparent in both Tpm2+/- and wild type (WT) mice. However, at 26-weeks old cataract was observed in the posterior part of the lens or annular opacity in Tpm2+/- mice, not in WT mice.
Tpm2+/- mice can be created using the CRISPR/Cas9 system. Tpm2 is involved in the progression of age-related cataract. The present results indicate that low levels of Tpm2 in the lens contribute the age-related cataract development. This is a useful model to study cataractogenesis and PCO.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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