- Technical Note
- Open Access
A pink mouse reports the switch from red to green fluorescence upon Cre-mediated recombination
© Hartwich et al.; licensee BioMed Central Ltd. 2012
Received: 10 January 2012
Accepted: 8 June 2012
Published: 14 June 2012
Targeted genetic modification in the mouse becomes increasingly important in biomedical and basic science. This goal is most often achieved by use of the Cre/loxP system and numerous Cre-driver mouse lines are currently generated. Their initial characterization requires reporter mouse lines to study the in vivo spatiotemporal activity of Cre.
Here, we report a dual fluorescence reporter mouse line, which switches expression from the red fluorescent protein mCherry to eGFP after Cre-mediated recombination. Both fluorescent proteins are expressed from the ubiquitously active and strong CAGGS promoter. Among the founders, we noticed a pink mouse line, expressing high levels of the red fluorescent protein mCherry throughout the entire body. Presence of mCherry in the living animal as well as in almost all organs was clearly visible without optical equipment. Upon Cre-activity, mCherry expression was switched to eGFP, demonstrating functionality of this reporter mouse line.
The pink mouse presented here is an attractive novel reporter line for fluorescence-based monitoring of Cre-activity. The high expression of mCherry, which is visible to the naked eye, facilitates breeding and crossing, as no genotyping is required to identify mice carrying the reporter allele. The presence of two fluorescent proteins allows in vivo monitoring of recombined and non-recombined cells. Finally, the pink mouse is an eye-catching animal model to demonstrate the power of transgenic techniques in teaching courses.
Spatiotemporally restricted genetic modification in the mouse is becoming an indispensable tool in biological research . Applications include analysis of essential genes, whose constitutive ablation results in embryonic/perinatal lethality, discrimination between primary and secondary effects, or mapping cell connectivity and cell fate [2–4]. The importance of targeted genetic modification is underlined by a recent global initiative to generate conditional alleles for all murine genes [5–7]. Most targeted genetic modifications make use of the Cre/loxP system. In this approach, a Cre-driver mouse line, expressing Cre recombinase of the phage P1 under a tissue or cell-type specific promoter, is crossed to mice with a loxP flanked (“floxed”) allele, which is recognized by Cre .
Several Cre-driver lines have been generated and each of them requires initial characterization of the spatial and temporal Cre expression pattern . A popular reporter line expresses LacZ from the ROSA26 locus upon Cre-mediated recombination , but its analysis is limited to fixed tissue. For in vivo observation of Cre-mediated recombination, several fluorescent reporter lines have been generated [11–15]. However, only few dual-reporter lines are available with different labels of recombined and non-recombined cells. Some of them combine a fluorescent protein with an enzyme (alkaline phosphatase or LacZ) [16–18], others combine two different fluorescent proteins [19, 20]. Here we present a novel Cre-reporter line with high and ubiquitous expression of two fluorescent proteins and which is easily recognized by its pink skin color.
Materials and methods
Transfection of HEK-293 cells
The plasmid pCAGGS_cherry_Intron_GFP was transiently transfected in HEK-293 cells with TurboFect (MBI Fermentas, St. Leon-Rot, Germany) according to the manufacturer´s instructions.
Generation and genotyping of transgenic mice
All animals used for these experiments were maintained on C57BL/6 J (Harlan Winkelmann, Borchen, Germany) and C57BL/6 N (Charles River, Sulzfeld, Germany) mixed genetic backgrounds. All protocols adhered to the German Animal Protection law and were approved by the local animal care and use committee (LAVES, Oldenburg). Protocols also followed the NIH guide for the care and use of laboratory animals.
Determination of transgene copy number
Transgene copy number was determined by semi-quantitative PCR, using the primers 5´-AAACATAACTTCGTATAATGTATGCTATACG-3´ and 5´-CGTAATGCAGAAGAAGACCATGG-3´ with the following conditions: denaturation for 5 min at 95 °C and 30 sec at 95 °C, annealing for 30 sec at 53 °C, 30 sec, synthesis for 30 sec at 72 °C and 5 min at 72 °C, 30 cycles with selfmade Taq-Polymerase. As a standard, we used the transgenic construct (~5 kb) in the following amounts: 0.2, 0.5, 1.0, and 1.5 pg. Band intensities were measured with ImageJ and compared to band intensities from PCR products of genomic DNA preparations from 5 different mice. PCR was done twice for standard and sample DNA. For copy number estimations, the factor 106 was used which reflects the size difference between the transgenic construct and the diploid mouse genome of ~ 5 Gb.
Tissue preparation and fluorescence microscopy
Mice were killed by gassing with CO2 and the organs immediately removed. Fluorescence of organs was either visualized with an UV lamp (Fluotest Original Hanau, Hanau, Germany) at 254 nm wavelength and a custom digital camera, or under an inverse microscope (Keyence, Neu-Isenburg, Germany).
Perfusion, slicing and immunohistochemistry
Mice were anesthetized with 70 mg/ml chloral hydrate (1 ml/100 g body weight) and perfused transcardially with phosphate-buffered saline (PBS) (130 mM NaCl, 7 mM Na2HPO4, 3 mM NaH2PO4, pH 7.4), followed by Zamboni´s solution (2 % paraformaldehyde, 15 % picric acid in 0.1 M phosphate buffer, pH 7.4). The brain was removed from the skull and incubated overnight in 30 % (w/v) sucrose/PBS. Coronal brainstem sections of 30 μm thickness were sliced using a microtome (MICROM GmbH, Walldorf, Germany), collected in 15 % (w/v) sucrose/PBS and washed twice in PBS and twice in PBST (PBS with 1 % (v/v) Triton X-100) (15 min each washing step). Sections were incubated for 1.5 h in blocking solution (2 % bovine serum albumin, 10 % goat serum and 0.3 % Triton X-100 in PBS, pH 7,4). Sections were transferred in blocking solution containing rabbit anti-GFP antibody (1:500, Invitrogen, Karlsruhe, Germany) and incubated overnight with agitation at 4 °C. After three washes with PBST (10 min each), sections were transferred into carrier solution (1 % bovine serum albumin, 1 % goat serum and 0.3 % Triton X-100 in PBS, pH 7,4) and treated with the secondary antibody anti-rabbit conjugated to Alexa Fluor 488 (1:500, Invitrogen). After 1.5 h with agitation at room temperature, slices were washed three times with PBS (10 min each) and mounted on glass slides with Vectashield Mounting Medium with DAPI (Vector Laboratories, Burlingame, USA). Photomicrographs were taken with an inverse microscope (Keyence) and the fluorescence was detected with the corresponding filter set.
In vivo slice preparation and imaging
Mice were killed by gassing with CO2 and 150 μm thick living brainstem slices were cut with a vibratome (MICROM GmbH). During the preparation, slices were incubated in 95 % O2 and 5 % CO2 aerated ACSF solution (118 mM NaCl, 1 mM NaH2PO4, 25 mM NaHCO3, 3 mM KCl, 1 mM MgCl2, 1.5 mM CaCl2, pH 7.4). After preparation, brainstem slices were examined with a TCS SL confocal microscope (Leica, Nussloch, Germany) using 40-fold magnification and a PL FLUOTAR objective (40.0x/0.7 NA).
Results and discussion
To generate a transgenic mouse line for in vivo cell labeling and the report of Cre-mediated recombination, we used the plasmid pCAGGS_cherry_Intron_GFP (Figure 1). This plasmid expresses the red fluorescent protein variant mCherry under the CAGGS promoter (chicken β-actin promoter with CMV enhancer), which is active in almost all tissues . Upon Cre-mediated expression, mCherry is deleted and eGFP expressed.
Pink colored mice
Whole organ fluorescence
This novel pink Cre-reporter mouse line represents a versatile tool in transgenic mouse research. A great advantage is the high expression level of the fluorescent protein mCherry, which allows easy recognition and separation of animals harboring the reporter allele from littermates without further analysis. The presence of two fluorescent proteins is useful for parallel in vivo monitoring of recombined and non-recombined cells, in vivo recordings, and analyses of cellular connectivity. Finally, this mouse line is well suited for teaching purposes. Its pink color can be used to easily demonstrate the power of transgenic technologies and crossed with an ubiquitously expressing Cre-driver, the color would disappear.
We thank K. Schönig and D. Bartsch, who kindly provided the plasmid and K. Dedek for help with the in vivo detection of fluorescent proteins. HH was supported by a stipend from the PhD program Hearing of the State of Lower Saxony. Financial support was provided by a grant from the EWE Stiftung to HGN. We thank Anja Feistner for excellent technical assistance.
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