Modified whole-mount in situ hybridization protocol for the detection of transgene expression in electroporated chick embryos

Abstract

Background: In vivo electroporation has been expensively used as an effective means of DNA transfer for analyzing gene function as well as gene regulation in developmental systems. In any of these two types of studies, the correct spatial and temporal expression of the electroporated transgene can only be accurately assessed by in situ hybridization. Methodology/Principal Findings: while analyzing transgene expression in electroporated chicken embryos, we verified that transgene riboprobes cross-hybridized with the exogenous plasmid DNA when embryos were processed by conventional whole mount in situ hybridization (WISH). Conclusion/Significance: Here we describe a mofication to the WISH protocol that is essential to prevent DNA cross-hybridization and to specifically detect transgene mRNA transcripts in electroporated embryos. Our optimized WISH procedure can be applied not only to electroporated chick embryos but also to other embryos or adult tissues that have been transfected with large amounts of reporter- or expression construct DNA. © 2008 Arede et al.