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Mol Biol Rep. 2012 Feb;39(2):1119-24. doi: 10.1007/s11033-011-0840-0. Epub 2011 Jun 17.

Transgenic mice with overexpression of mutated human optineurin(E50K) in the retina.

Author information

1
Department of Ophthalmology, Second Affiliated Hospital of Harbin Medical University, No. 246 Xuefu street, Nangang district, Heilongjiang, Harbin 150086, China.

Abstract

In the present work, Site-directed mutagenesis to insert the Glu50Lys amino acid substitution was achieved by PCR using plasmid pBluescript-OPTN. Mutated human OPTN(E50K) gene-driven mouse c-kit promoter was constructed and confirmed by endonuclease digestion and sequence analysis. Transgenic mice were generated via the microinjection method. PCR and DNA dot blot were used to screen the positive transgenic mice. RT-PCR analyzed the RNA level and location of mutated human OPTN(E50K) mRNA expression in transgenic mice. Western blot and immunohistochemistry were used to detect the level and location of mutated human OPTN(E50K) expression in transgenic mice. A transgenic mouse model with overexpression of mutated human OPTN(E50K) in retina was successfully established. The transgene was integrated and transmitted into the chromosome of transgenic mice. Mutated human OPTN(E50K) gene was controlled by c-kit promoter and expressed in the retina in mice. Mutated human OPTN(E50K) in transgenic mice was higher than that of wild type C57BL/6J mice. Our studies had provided a new transgenic model for investigating the molecular properties of mutated human OPTN(E50K).

PMID:
21681420
DOI:
10.1007/s11033-011-0840-0
[Indexed for MEDLINE]

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