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Zusammenfassung:
The chick model has been instrumental in illuminating genes that regulate early vertebrate development and pattern formation. Targeted ectopic gene expression is critical to dissect further the complicated gene interactions that are involved. In an effort to develop a consistent method to ectopically introduce and focally express genes in chick mesoderm, we evaluated and optimized several gene delivery methods, including implantation of 293 cells laden with viral vectors, direct adenoviral injection, and electroporation (EP). We targeted the mesoderm of chick wing buds between stages 19 and 21 (Hamburger and Hamilton stages) and used beta-galactosidase and green fluorescent protein (GFP) to document gene transfer. Expression constructs using the cytomegalovirus (CMV) promoter, the beta-actin promoter, and vectors with an internal ribosomal entry sequence linked to GFP (IRES-GFP) were also compared. After gene transfer, we monitored expression for up to 3 days. The functionality of ectopic expression was demonstrated with constructs containing the coding sequences for Shh, a secreted signaling protein, or Hoxb-8, a transcription factor, both of which can induce digit duplication when ectopically expressed in anterior limb mesoderm. We identified several factors that enhance mesodermal gene transfer. First, the use of a vector with the beta-actin promoter coupled to the 69% fragment of the bovine papilloma virus yielded superior mesodermal expression both by markers and functional results when compared with several CMV-driven vectors. Second, we found the use of mineral oil to be an important adjuvant for EP and direct viral injection to localize and contain vector within the mesoderm at the injection site. Lastly, although ectopic expression could be achieved with all three methods, we favored EP confined to the mesoderm with insulated microelectrodes (confined microelectroporation-CMEP), because vector construction is rapid, the method is efficient, and results were consistent and reproducible.
