Understanding gene functions and disease mechanisms: Phenotyping pipelines in the German Mouse Clinic

Fuchs, Helmut; Aguilar-Pimentel, Juan Antonio; Amarie, Oana V.; Becker, Lore; Calzada‐Wack, Julia; Cho, Yi Li; Garrett, Lillian; Hölter, Sabine M.; Irmler, Martin; Kistler, Martin; Kraiger, Markus; Mayer‐Kuckuk, Philipp; Moreth, Kristin; Rathkolb, Birgit; Rozman, Jan; Buttkus, Patricia da Silva; Treise, Irina; Zimprich, Annemarie; Gampe, Kristine; Hutterer, Christine; Stöger, Claudia; Leuchtenberger, Stefanie; Maier, Holger R.; Miller, Manuel; Scheideler, Angelika; Wu, Moya; Beckers, Johannes; Bekeredjian, Raffi; Brielmeier, Markus; Busch, Dirk H.; Klopstock, Thomas; Ollert, Markus; Schmidt‐Weber, Carsten B.; Stöger, Tobias; Wolf, Eckhard; Wurst, Wolfgang; Yildirim, Ali Önder; Zimmer, Andreas; Gailus‐Durner, Valérie; Angelis, Martin Hrabé de

doi:10.1016/j.bbr.2017.09.048

Cited by 34 publications

(36 citation statements)

References 56 publications

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“…Health status was confirmed to be specific pathogen-free according to FELASA recommendations. Using the platform established at the GMC, we performed a primary phenotypic analysis of a total of 60 (15 mice each of each genotype and sex) [27–29] exactly as described in the Additional file 6.…”

Section: Methodsmentioning

confidence: 99%

Claudin-12 is not required for blood–brain barrier tight junction function

Dias

Coisne

Baden

et al. 2019

Fluids Barriers CNS

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Background The blood–brain barrier (BBB) ensures central nervous system (CNS) homeostasis by strictly controlling the passage of molecules and solutes from the bloodstream into the CNS. Complex and continuous tight junctions (TJs) between brain endothelial cells block uncontrolled paracellular diffusion of molecules across the BBB, with claudin-5 being its dominant TJs protein. However, claudin-5 deficient mice still display ultrastructurally normal TJs, suggesting the contribution of other claudins or tight-junction associated proteins in establishing BBB junctional complexes. Expression of claudin-12 at the BBB has been reported, however the exact function and subcellular localization of this atypical claudin remains unknown. Methods We created claudin-12-lacZ-knock-in C57BL/6J mice to explore expression of claudin-12 and its role in establishing BBB TJs function during health and neuroinflammation. We furthermore performed a broad standardized phenotypic check-up of the mouse mutant. Results Making use of the lacZ reporter allele, we found claudin-12 to be broadly expressed in numerous organs. In the CNS, expression of claudin-12 was detected in many cell types with very low expression in brain endothelium. Claudin-12lacZ/lacZ C57BL/6J mice lacking claudin-12 expression displayed an intact BBB and did not show any signs of BBB dysfunction or aggravated neuroinflammation in an animal model for multiple sclerosis. Determining the precise localization of claudin-12 at the BBB was prohibited by the fact that available anti-claudin-12 antibodies showed comparable detection and staining patterns in tissues from wild-type and claudin-12lacZ/lacZ C57BL/6J mice. Conclusions Our present study thus shows that claudin-12 is not essential in establishing or maintaining BBB TJs integrity. Claudin-12 is rather expressed in cells that typically lack TJs suggesting that claudin-12 plays a role other than forming classical TJs. At the same time, in depth phenotypic screening of clinically relevant organ functions of claudin-12lacZ/lacZ C57BL/6J mice suggested the involvement of claudin-12 in some neurological but, more prominently, in cardiovascular functions.

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Section: Methodsmentioning

confidence: 99%

Claudin-12 is not required for blood–brain barrier tight junction function

Dias

Coisne

Baden

et al. 2019

Fluids Barriers CNS

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“…The Mettl5 KO mouse model was derived using CRISPR/Cas technology and systematically characterized in the German Mouse Clinic phenotyping screen as described previously (Gailus-Durner et al 2005;Fuchs et al 2018). For the single-guide RNA (sgRNA) selection in vitro, two 20-bp sgRNA sequences targeting Mettl5 exon1 (gene ID 75422) were screened on the Web site http ://crispor.tefor.net and synthesized by using the in vitro transcription EnGen Kit (NEB E3322S).…”

Section: Crispr/cas9 Gene Editing In Micementioning

confidence: 99%

The rRNA m⁶A methyltransferase METTL5 is involved in pluripotency and developmental programs

et al. 2020

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Covalent chemical modifications of cellular RNAs directly impact all biological processes. However, our mechanistic understanding of the enzymes catalyzing these modifications, their substrates and biological functions, remains vague. Amongst RNA modifications N 6-methyladenosine (m 6 A) is widespread and found in messenger (mRNA), ribosomal (rRNA), and noncoding RNAs. Here, we undertook a systematic screen to uncover new RNA methyltransferases. We demonstrate that the methyltransferase-like 5 (METTL5) protein catalyzes m 6 A in 18S rRNA at position A 1832. We report that absence of Mettl5 in mouse embryonic stem cells (mESCs) results in a decrease in global translation rate, spontaneous loss of pluripotency, and compromised differentiation potential. METTL5-deficient mice are born at non-Mendelian rates and develop morphological and behavioral abnormalities. Importantly, mice lacking METTL5 recapitulate symptoms of patients with DNA variants in METTL5, thereby providing a new mouse disease model. Overall, our biochemical, molecular, and in vivo characterization highlights the importance of m 6 A in rRNA in stemness, differentiation, development, and diseases.

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“…Homozygous CRN2 knock-out mice and wild-type control littermates were subjected to a systematic, comprehensive phenotyping screen at the German Mouse Clinic at the Helmholtz Zentrum München (http://www.mouseclinic.de) as described previously (Fuchs et al, 2018(Fuchs et al, , 2011(Fuchs et al, , 2012Gailus-Durner et al, 2005). This screen covers a broad range of parameters in the areas of allergy, behaviour, cardiovascular function, clinical chemistry, dysmorphology, energy metabolism, eye analysis and vision, hematology, immunology, neurology, and pathology.…”

Section: Phenotypic Analysis In the German Mouse Clinicmentioning

confidence: 99%

CRN2 binds to TIMP4 and MMP14 and promotes perivascular invasion of glioblastoma cells

Solga

Behrens

Ziemann

et al. 2019

European Journal of Cell Biology

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CRN2 is an actin filament binding protein involved in the regulation of various cellular processes including cell migration and invasion. CRN2 has been implicated in the malignant progression of different types of human cancer. We used CRN2 knock-out mice for analyses as well as for crossbreeding with a Tp53/Pten knock-out glioblastoma mouse model. CRN2 knock-out mice were subjected to a phenotyping screen at the German Mouse Clinic. Murine glioblastoma tissue specimens as well as cultured murine brain slices and glioblastoma cell lines were investigated by immunohistochemistry, immunofluorescence, and cell biological experiments. Protein interactions were studied by immunoprecipitation, pull-down, and enzyme activity assays. CRN2 knock-out mice displayed neurological and behavioural alterations, e.g. reduced hearing sensitivity, reduced acoustic startle response, hypoactivity, and less frequent urination. While glioblastoma mice with or without the additional CRN2 knock-out allele exhibited no significant difference in their survival rates, the increased levels of CRN2 in transplanted glioblastoma cells caused a higher tumour cell encasement of murine brain slice capillaries. We identified two important factors of the tumour microenvironment, the tissue inhibitor of matrix metalloproteinase 4 (TIMP4) and the matrix metalloproteinase 14 (MMP14, synonym: MT1-MMP), as novel binding partners of CRN2. All three proteins mutually interacted and co-localised at the front of lamellipodia, and CRN2 was newly detected in exosomes. On the functional level, we demonstrate that CRN2 increased the secretion of TIMP4 as well as the catalytic activity of MMP14. Our results imply that CRN2 represents a pro-invasive effector within the tumour cell microenvironment of glioblastoma multiforme.

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Understanding gene functions and disease mechanisms: Phenotyping pipelines in the German Mouse Clinic

Cited by 34 publications

References 56 publications

Claudin-12 is not required for blood–brain barrier tight junction function

Claudin-12 is not required for blood–brain barrier tight junction function

The rRNA m⁶A methyltransferase METTL5 is involved in pluripotency and developmental programs

CRN2 binds to TIMP4 and MMP14 and promotes perivascular invasion of glioblastoma cells

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Understanding gene functions and disease mechanisms: Phenotyping pipelines in the German Mouse Clinic

Cited by 34 publications

References 56 publications

Claudin-12 is not required for blood–brain barrier tight junction function

Claudin-12 is not required for blood–brain barrier tight junction function

The rRNA m6A methyltransferase METTL5 is involved in pluripotency and developmental programs

CRN2 binds to TIMP4 and MMP14 and promotes perivascular invasion of glioblastoma cells

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Product

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The rRNA m⁶A methyltransferase METTL5 is involved in pluripotency and developmental programs