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F. Maurer, T. Pedrazzini, C. Kostic, Y. Arsenijevic, A. Bessero, F.L. Munier, D.F. Schorderet; Lentivirus-mediated Generation of Transgenic Mice Expressing the Human R555W BIGH3 Protein (Groenouw Type I Mutation) . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3849.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To generate mouse models of BIGH3-linked corneal dystrophies. Methods: A human cDNA encoding the R555W BIGH3 protein (Groenouw type I mutation) was placed under the control of the murine phosphoglycerate kinase 1 (PGK1) promoter and inserted into a self-inactivating lentiviral vector. To increase the level of expression, the woodchuck hepatitis virus posttranscriptional regulatory element (WRE) was inserted downstream of BIGH3 cDNA. Viruses were pseudotyped with the vesicular stomatitis virus glycoprotein (VSVG) and concentrated by ultracentrifugation. One- or two-cell mouse embryos were transduced in vitro by co-incubation with lentiviral particles (Lois et al. Science, 2002). These embryos were transferred into foster mice and carried to term. Screening of founders were performed by PCR and Southern blot analyses. Expression was tested by RT-PCR and Western blotting. Results: Five transfers into foster mothers were performed with 25 transduced embryos each. In a total of 25 offspring, we identified eight transgenic mice, carrying various copy numbers of the complete R555W BIGH3 construct. Mice are viable and fertile and do not present obvious phenotypic alterations. Preliminary expression analyses suggest that the transgene is properly transcribed in the founders. Complete physical and ophthalmologic examinations of these mice are in progress and will be discussed. Conclusions: The rapid and efficient lentivirus-mediated production of transgenic mice carrying BIGH3 should prove useful to investigate the physiological relevance of a number of mutations in the BIGH3 gene, in particular in regard to their role in the development of corneal dystrophies.
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