The Cre/loxP system is commonly used to generate conditional knockout mice. A
large number of mouse cre lines have been developed and each of them requires
characterisation of the spatial and temporal pattern of cre expression (Nagy and Mar,
2001). This is normally achieved using cre reporter mouse lines. Nowadays, several
double-fluorescent cre reporter mouse lines are available which express green
fluorescent protein (GFP) in recombined cells and a complementary red fluorescent
protein (RFP) in non-recombined cells (Muzumdar et al., 2007; Hartwich et al., 2012).
During this work I cloned a transgenic vector for production of a double-fluorescent
bioluminescent reporter mouse that expresses Venus prior to cre recombination and
Fluc2 and mCherry after cre recombination. The first step was to design the construct
with the reporter genes, the stop signal which separates them and the loxP sites
placed in a strategic manner. At this point it was important to erase all undesired
recognition sites of restriction enzymes to avoid disruption of the construct at any of
the following cloning steps. Another problem was the choice of a reliable stop signal
to ensure that Fluc2 and mCherry only get expressed in the presence of cre
In a series of cloning steps the reporter construct was shuttled into different
expression vectors for pronuclear injection (pCAG-Venus3xStop-Fluc2-P2AmCherry)
and transposon mediated transgenesis (pTN-Venus3xStop-Fluc2-P2AmCherry).
The vector for pronuclear injection was tested by transient transfection of human
embryonic kidney (HEK) 293T cells. I transfected different numbers of cells with
either pCAG- Venus3xStop-Fluc2-P2A-mCherry only or with pCAG- Venus3xStop-
Fluc2-P2A-mCherry plus pCAGGS-NLS-Cre.
The cells were analysed under a Zeiss Axiovert 200 fluorescent microscope 48 hours
after the transfection. I observed strong Venus and minimal mCherry expression in
cells transfected with pCAG-Venus3xStop-Fluc2-P2A-mCherry only in contrast to
weak Venus and strong mCherry expression in cells co-transfected with pCAGVenus3xStop-
Fluc2-P2A-mCherry and pCAGGS-NLS-Cre. In order to detect Fluc2 expression I added D-luciferin (15 mg/ml) at a final
concentration of 150 μg/ml to the cells and measured bioluminescence after 0, 5 and
10 minutes with a Xenogen IVIS® 50 imager. In cells co-transfected with pCAGVenus3xStop-
Fluc2-P2A-mCherry and pCAGGS-NLS-Cre I observed a more than
60-fold increase of the bioluminescent signal compared to the cells transfected with
pCAG- Venus3xStop-Fluc2-P2A-mCherry only. Both results indicate that the vector
works properly in vitro and cre mediated recombination occurred.