Targeted gene expression in the chicken eye by in ovoelectroporation

[摘要] Purpose: The chicken embryo lens is a classical model system fordevelopmental and cell biology studies. To understand the molecularmechanisms that underlie the morphological changes that occur duringlens development, it is important to develop an effective gene transfermethod that permits the analysis of gene functions in vivo. In ovoelectroporation has been successfully used for introducing DNA intoneural and mesenchymal tissues of chicken embryos. In this study, weexplored the possibility of using this technique to manipulate geneexpression in lens epithelial and fiber cells, as well as in other cellsof the chicken eye.Methods: Two DNA constructs were used in this study. pCAX contains achicken β-actin promoter fused to the CMV IE enhancer to driveenhanced green fluorescent protein (EGFP) expression. pMES-cNf2 uses thesame chimeric promoter to drive the expression of the chickenneurofibromatosis 2 (cNf2) and EGFP proteins in the same cell. PlasmidDNA was injected into the lumen of the lens vesicle in chicken embryosat stage 15. For corneal epithelial and retinal cell electroporation,DNA was placed near the surface ectoderm in the eye region or injectedinto the vitreous cavity, respectively. Electroporation was performedwith one electrode above the eye and the other underneath the head ofthe embryo. Chicken embryos were harvested at different time points forEGFP expression analysis by immunohistochemistry.5-bromo-2'-deoxyuridine (BrdU) incorporation assays were used toevaluate the effects of cNf2 on lens epithelial cell proliferation.Results: A strong EGFP signal can be detected in lens cells 4 hafter electroporation. The transfected cells maintain high levels ofEGFP expression for at least 5 days. Overexpressing cNf2 in lensepithelial cells significantly inhibits cell proliferation. Ectopicexpression of EGFP in corneal epithelial and retinal cells was alsoachieved by in ovo electroporation.Conclusions: We have demonstrated that exogenous DNA can beeffectively introduced into lens, corneal and retinal cells in theliving embryo by in ovo electroporation. In comparison to viralinfection and transgenic mouse approaches, in ovo electroporation offersan easier and quicker way to manipulate gene expression during embryonicdevelopment. This technique will be a useful tool for exploring themolecular mechanisms of lens and eye development.

[学科分类] 生物化学/生物物理