Background
The ease of manipulating its genome has helped establish the mouse as the premier model organism for mammalian genetic studies. Both directed and random mutagenesis approaches, including the technologies of transgenesis and gene targeting in ES cells, have become commonplace. Genetically manipulated mice, often incorporating gene-based reporters, are frequently being used to model and understand mammalian development and disease processes. Fluorescent protein reporters currently represent a superior alternative to other gene-based reporters such as the bacterial lacZ or human placental alkaline phosphatase in that their visualization is non-invasive, and as such does not require chromogenic substrates. Fluorescence can be monitored in real-time in vivo and in situ, and has the added advantage in that it can be quantified. The prototype fluorescent protein reporter is green fluorescent protein (GFP, reviewed in [3]), which is derived from the bioluminescent jellyfish Aequorea Victoria [1].
Green fluorescent variants of wild type GFP (wtGFP) with improved thermostability and fluorescence emission, including enhanced green fluorescent protein (EGFP, [4]), and mMGFP [5], have gained popularity for use in mice. Recently however, several additional mutants of wtGFP, with altered excitation and emission spectral profiles (i.e. fluoresce in colors other than green), as well as improved thermostability and fluorescence, have been described [4,6].
Dual color imaging of several GFP variant proteins is a very attractive prospect and has become a standard procedure in cell biology [7,8]. It has also recently been demonstrated in a subset of cells within the central nervous system (CNS) of mice using both transgenic and viral infection-based approaches [9,10]. We therefore wished to investigate the feasibility of non-invasive multiple reporter imaging in mice, and as a first step we wanted to ascertain whether we could establish viable and fertile mice with widespread expression of GFP variant reporters.