Identification of Undescribed Relb Expression Domains in the Murine Brain by New Relb:cre-katushka Reporter Mice
Authors
Christian Engelmann1, Marc Riemann1, Swen Carlstedt1,2, Randy Grimlowski1,3, Nico Andreas1,4, Ievgen Koliesnik1,5, Elke Meier1, Phillip Austerfield1, Ronny Haenold1,#
1 Leibniz Institute on Aging − Fritz-Lipmann-Institute (FLI), Beutenbergstrasse 11, 07745 Jena, Germany
2 current address: Jena University Hospital, Institute of Biochemistry II, 07747 Jena, Germany
3 current address: Max Planck Institute for Molecular Biomedicine, Department of Vascular Cell Biology, 48149 Münster, Germany
4 current address: Jena University Hospital, Institute of Immunology, 07743 Jena, Germany
5 current address: Stanford University, School of Medicine, Division of Infectious Diseases and Geographic Medicine, 94305-5107 Stanford, CA, USA
# Corresponding author: Ronny Haenold, Leibniz Institute on Aging − Fritz-Lipmann-Institute (FLI), Beutenbergstrasse 11, 07745 Jena, Germany; Phone: +49-3641-656817; E-mail: ronny.haenold@leibniz-fli.de
Abstract
Background: Noncanonical NF-κB signaling through activation of the transcription factor RelB acts as key regulator of cell lineage determination and differentiation in various tissues including the immune system. To elucidate temporospatial aspects of Relb expression, we generated a BAC transgenic knock-in mouse expressing the fluorescent protein Katushka and the enzyme Cre recombinase under control of the murine Relb promoter (RelbCre-Kat mice).
Results: Co-expression of katushka and Relb in fibroblast cultures and tissues of transgenic mice revealed highly specific reporter functions of the transgene. Crossing RelbCre-Kat mice with ROSA26R reporter mice that allow for Cre-mediated consecutive β-galactosidase or YFP synthesis identified various Relb expression domains in perinatal and mature mice. Besides thymus and spleen, highly specific expression patterns were found in different neuronal domains, as well as in other non-immune organs including skin, skeletal structures and kidney. De nove Relb expression in the mature brain was confirmed in conditional knockout mice with neuro-ectodermal Relb deletion.
Conclusion: Our results demonstrate the usability of RelbCre-Kat reporter mice for the detection of de novo and temporarily restricted Relb expression including cell and lineage tracing of Relb expressing cells. Relb expression during mouse embryogenesis and at adulthood suggests, beyond immunity, important functions of this transcription factor in neurodevelopment and CNS function.
This image gallery is a collection of histological serial sections from a 2-day-old RelbCre-Kat;ROSA26R reporter mouse to depict tissue-specific and cellular Relb expression patterns in the whole organism.
Methods
Transgenic mice
To create a high-fidelity Relb reporter gene, a bacterial artificial chromosome (BAC) of 93 kb (bMQ-258k08) containing the complete Relb locus with 45 kb of upstream region was modified by introducing the gene coding for a fusion protein of Cre recombinase and Katushka, both linked by a self-cleaving P2A peptide (Kim, Lee et al. 2011), into the first exon of the Relb gene just downstream of the translation initiation codon (Relb:cre–katushka). The vector was injected into pro-nuclei from fertilized C57BL/6J oocytes. Three founder mice that transmitted the transgene to the progeny were analyzed for transgene expression. In one of the three founder lines transgene expression satisfactorily reflected that of endogenous Relb (termed RelbCre-Kat mice). Transgenic mice were generated by PolyGene (Rümlang, Switzerland). For cell tracing of Relb expression in developing and mature organs, RelbCre-Kat mice were crossed with ROSA26-LacZ mice (Soriano 1999) that allow for irreversible expression of β-galactosidase after Cre-mediated excision of a loxP-flanked STOP sequence (Fig. 1). Double-transgenic mice heterozygous for RelbCre-Kat and ROSA26-LacZ transgenes (termed RelbCre-Kat;LacZ mice), and single or non-transgenic controls were used for analysis of β-galactosidase activity.
Figure 1: Scheme of reporter protein expression in double-transgenic RelbCre-Kat;Rosa26R reporter mice.
In RelbCre-Kat reporter mice, expression of the Cre-P2A-Katushka transgene is driven by the murine Relb promoter.
Inducible expression of cre recombinase leads to constitutive expression
of β-galactosidase (detected by X-gal staining) in RelbCre-Kat;LacZ double transgenic mice.
Histology
For whole body sections of postnatal mice (P2), animals were sacrificed and snap-frozen in methyl-butane. Mice were embedded in Tissue Tec for preparation of serial sagittal cryo-sections with a slice thickness of 16 µm at a distance of approximately 100 µm on a cryostat-microtome (Leica CM3000; Wetzlar, Germany) (Fig. 2). Tissue sections were postfixed for 5 min in 4% buffered paraformaldehyde (PFA, pH 7.4), rinsed in PBS and incubated in X-gal staining solution (Roche, Mannheim, Germany) for 24 hrs at 37 ̊C. Section were counterstained with eosin to depict anatomical structures. For anatomical orientation, sections 1, 8, 17, and 35 were solely stained with H&E. Sections were scanned under transmission light using an Olympus Virtual Slide microscope at 20x magnification (numerical aperture [NA] 0.75) or 40x (NA 0.9) magnification. Focus plane and stitching of the tiles were set automatically. Scanned tiles were aligned to obtain the final images.
Figure 2: Preparation of whole body cryo-sections from postnatal mice (shown for P7).
(A) Sacrificed animals were snap-frozen and immediately mounted on a cryostat-microtome.
Mice were embedded in layers with Tissue Tec and 16 µm cryo-sections were prepared using
a standard microtome razor blade (B). Quality of sections was validated by H&E staining (C).
Images
Please select an image
-
Section 1Hematoxylin & Eosin
-
Section 2X-Gal & Eosin
-
Section 3X-Gal & Eosin
-
Section 4X-Gal & Eosin
-
Section 5X-Gal & Eosin
-
Section 6X-Gal & Eosin
-
Section 7X-Gal & Eosin
-
Section 8Hematoxylin & Eosin
-
Section 9X-Gal & Eosin
-
Section 10X-Gal & Eosin
-
Section 11X-Gal & Eosin
-
Section 12X-Gal & Eosin
-
Section 13X-Gal & Eosin
-
Section 14X-Gal & Eosin
-
Section 15X-Gal & Eosin
-
Section 16X-Gal & Eosin
-
Section 17Hematoxylin & Eosin
-
Section 18X-Gal & Eosin
-
Section 19X-Gal & Eosin
-
Section 20X-Gal & Eosin
-
Section 21X-Gal & Eosin
-
Section 22X-Gal & Eosin
-
Section 23X-Gal & Eosin
-
Section 24X-Gal & Eosin
-
Section 25X-Gal & Eosin
-
Section 26X-Gal & Eosin
-
Section 27X-Gal & Eosin
-
Section 28X-Gal & Eosin
-
Section 29X-Gal & Eosin
-
Section 30X-Gal & Eosin
-
Section 31X-Gal & Eosin
-
Section 32X-Gal & Eosin
-
Section 33X-Gal & Eosin
-
Section 34X-Gal & Eosin
-
Section 35Hematoxylin & Eosin
-
Section 36X-Gal & Eosin
-
Section 37X-Gal & Eosin
-
Section 38X-Gal & Eosin
-
Section 39X-Gal & Eosin
-
Section 40X-Gal & Eosin
-
Section 41X-Gal & Eosin
-
Section 42X-Gal & Eosin
-
Section 43X-Gal & Eosin
-
Section 44X-Gal & Eosin
-
Section 45X-Gal & Eosin
-
Section 46X-Gal & Eosin
-
Section 47X-Gal & Eosin
Credits
This work is dedicated to Professor Dr. Falk Weih (1959–2014). We thank Dr. Lucien Frappart (Leibniz Institute on Aging) for histological advice. The Histology Service of the FLI is gratefully acknowledged for technical support. This work was supported by the Leibniz Graduate School on Ageing and Age-Related Diseases (LGSA) (to C.E.), as well as by grants from the German Research Association (DFG grant number WE 2224/6-2 to M.R.) and the VELUX STIFTUNG, Switzerland (to R.H.). The FLI is a member of the Leibniz Association and is financially supported by the Federal Government of Germany and the State of Thuringia.
