A new inducible transgenic mouse model for C9orf72-associated GGGGCC repeat expansion

Renate K. Hukema1*

Fréderike W. Riemslagh1,2 *

Shamiram Melhem2

Herma C. van der Linde1

Lies-Anne W.F.M. Severijnen1

Dieter Edbauer3

Alex Maas4

Nicolas Charlet-Berguerand5

Rob Willemsen1,#

John C. van Swieten2,6,#

1 Department of Clinical Genetics, Erasmus Medical Center, 3015 CE Rotterdam, The Netherlands

2 Department of Neurology, Erasmus Medical Center, 3015 CE Rotterdam, The Netherlands

3 German Center for Neurodegenerative Diseases, 81337, Munich, Germany

4 Department of Cell Biology, Erasmus Medical Center, 3015 CE Rotterdam, The Netherlands

5 Department of Neurobiology and Genetics, IGBMC, INSERM U964, CNRS UMR7104, University of Strasbourg, Illkirch, France

6 Department of Neurology, Neuroscience Campus Amsterdam, 1007 MB Amsterdam, The Netherlands

*,# Authors contributed equally to this work

Supplementary materials and methods

Construct and mice

The pCMV-RL plasmid (Promega) was modified to express eGFP (enhanced green fluorescent protein) under the control of the tetracycline responsive promoter element (TRE). Utilizing oligonucleotide poly-linkers the CMV promoter in pRL-CMV was replaced with the TRE promoter from the pTRE2 vector (Clontech). The eGFP reporter from the eGFPn-1 vector (Clontech) was cloned downstream of the chimeric intron followed by a SV40 poly A signal. Immediately downstream of the TRE promoter and upstream of eGFP the 80GGGGCC repeat was cloned. The 80GGGGCC repeat was constructed by serial ligation of 4 artificial 20GGGGCC repeats (kind gift of Nicolas Charlet-Berguerand). Transgenic mice were generated by injecting linearized construct into oocytes of Janvier.c57bl6 mice. We used the founders that gave optimal expression, combined with good breeding results. TRE-80GGGGCC-eGFP mice were crossed with the hnRNP-rtTA driver line[1] and bigenic mice were treated with doxycycline (dox) directly after weaning at an age of approximately 3-4 weeks. Dox drinking water contained 2 mg/ml doxycycline hyclate (Sigma) in 5% sucrose (Sigma) and was refreshed every 2-3 days. All animal experiments were conducted with the permission of the local animal welfare committee (DEC EMC2912).


For genotyping toe clips were incubated overnight at 55°C in 300µl tailmix (100mM NaCl, 10mM Tris, 15mM EDTA, 0.5% SDS) and 200µg Proteinase K (Roche). DNA is isolated by first adding 100µl 6M NaCl, mixing and centrifuging the tube 10 min at 13000rpm. The supernatant was transferred to a new tube and 1ml 100% ethanol was added to precipitate the DNA, the tubes were centrifuged again for 10 min at 13000rpm. The supernatant was removed and 500µl 70% ethanol was added to wash the pellet, which was also centrifuged and then left to dry after removing the supernatant. The DNA pellet was dissolved in 100µl milli-Q water. The TRE transgene was amplified using forward primer 5’-gcttagatctctcgagtttac-3’ and reverse primer 5’-ATGGAGGTCAAAACAGCGTG-3’. The rtTA transgene was amplified using forward primer 5’-CAGCAGGCAGCATATCAAGGT-3’ and reverse primer 5’-GCCGTGGGCCACTTTACAC-3’ in the following PCR mix (1x PCR buffer, 2.0 mM MgCl2, 0.01% W1, 0.25 mM of each dNTP and dH2O). To quantify the number of C9orf72 repeat expansions repeats in the TRE-GGGGCC-eGFP mice, PCR mix and program according to Hukema and Oostra (2013)[2] were used in combination with adjusted primers: forward primer 5’-CGGGTCGAGTAGGCGTGTAC-3’ and reverse primer 5’-CCATGATATAGACGTTGTGGC-3’.


Tissues were fixed overnight in 4% paraformaldehyde and embedded in paraffin according to standard protocols. Sections (6µm) were cut using a Microm HM 335 E microtome and placed on SuperFrost slides (Menzel-Gläser, Braunschweig, Germany). Deparaffinisation was followed by antigen retrieval using microwave treatment in 0.01M sodium citrate. Endogenous peroxidase activity was blocked and immunostaining was performed overnight at 4°C using mouse anti-GFP antibody (Roche, Almere, The Netherlands), anti-ubiquitin antibody (Dako, Heverlee, Belgium), anti-TDP-43 rabbit antibody (ProteinTech Group, Manchester, United Kingdom) and mouse anti-poly-GA antibody (kind gift of D. Edbauer). Antigen-antibody complexes were visualized by incubation with DAB substrate (Dako) after incubation with Brightvision poly-HRP-linker (Immunologic). Slides were counterstained with Mayer’s haematoxylin and mounted with Entellan (Merck Millipore International). The slides were then left to dry in the fume hood for an hour and thereafter put in at 37°C overnight. Pictures were taken by using an Olympus BX40 microscope (Olympus, Melville, U.S.A.).

Supplementary Material and Methods References:

1. Katsantoni EZ, Anghelescu NE, Rottier R, Moerland M, Antoniou M, de Crom R, Grosveld F, Strouboulis J (2007) Ubiquitous expression of the rtTA2S-M2 inducible system in transgenic mice driven by the human hnRNPA2B1/CBX3 CpG island. BMC Dev Biol 7:108. doi:1471-213X-7-108 [pii]


2. Hukema RK, Oostra BA (2013) The CGG repeat and the FMR1 gene. Methods Mol Biol 1010:155-176. doi:10.1007/978-1-62703-411-1_11