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Publication type: Baccalaureate Thesis

Year: 2013

Author(s): Niedermayer, Constantin

Title: Cloning and in vitro Verification of a Fluorescent / Bioluminescent Reporter Construct for the production of an improved Cre-Reporter Mouse.

Other title: Klonierung und in vitro Verifikation eines Fluoreszenz/ Bioluminezenz Reporterkonstrukts für die Generierung einer verbesserten Cre-Reporter Maus

Source: Bakkalaureatsarbeit, Vet. Med. Univ. Wien, pp. 50.


Advisor(s):

Fink Dieter

Reviewer(s):
Müller Mathias

Vetmed Research Units:
Institute of Laboratory Animal Science


Graduation date: 02.08.13


Abstract:
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 recombinase. 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.


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