Embryonic stem (ES) cells hold significant potential for studying the early developmental pathways of tissue differentiation and function. However, the identification and isolation of pure cell populations has been hampered by the heterogeneity of ES cell cultures and a paucity of robust genetic markers in ES cell lines. One popular approach for identifying gene expression in living tissues by microscopy or flow cytometry 
is with fluorescent proteins. In addition to the wide variety of colors, fluorescent markers with long [e.g., enhanced and emerald GFPs (EmGFP)] 
or short (e.g., destabilized GFP) 
half-lives are available.
Many current reporters achieve cell specificity by linking a small promoter fragment to a protein/reporter fusion, replacing an entire open reading frame with a reporter, or using an internal ribosomal entry sites (IRES) sequence to drive reporter expression. These constructs are not ideal for poorly characterized or large promoter regions. In addition, polycistronic constructs with IRES sequences can display differential expression of the individual cistrons 
, making it difficult to directly correlate promoter function to fluorescence levels.
Bacterial artificial chromosomes (BACs) of up to 300 kb have been used to create reporter constructs and transgenic animals 
. BAC reporters are capable of carrying large promoter and enhancer regions within a single construct. The long BAC arms also function to decrease integration-site effects in transgenic cell lines or mice. Rapid and efficient BAC modification techniques have been recently introduced 
, and methods for creating GFP-based mouse BAC reporters for use in ES cells have also been described 
Ideally, reporter constructs should be easily created with different combinations of fluorophores and bacterial/eukaryotic selection markers linked to the same promoter region. This is particularly important when the reporters are to be used in different cell types or in cells from different species, since the efficiencies of the promoters driving selection markers vary 
. In addition, peptide sequences added to fluorophores, such as GFP, may affect protein stability 
or organelle localization (e.g., if a signal sequence is attached), suggesting that a uniform fluorophore molecule may be beneficial when quantitative comparisons between different reporter lines are desired. Finally, modifications to the 5′ un-translated region and endogenous ATG start site may be undesirable since both of these regions are subject to chromatin modification 
for transcriptional regulation.
The method presented here addresses these concerns and uses high-efficiency recombination techniques to minimize the need for unique reagents.