Analysis of Gene Functions In Vivo by Generation and Characterization of Genetically Modified Mice

Project leader
Docent Raija Soininen, Ph.D.
Oulu Center for Cell-Matrix Research, Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu

Background and Significance

Genetically modified (GM) mice are important tools in analysis of genes and gene products in vivo. They provide valuable information about gene functions and serve as models of human diseases. The BCO Transgenic core facility provides services in generation, archiving, re-derivation, and analysis of genetically modified mice (www.oulu.fi/biocenter/tgcore). Consultation, education of researchers, and following and being involved in the development and set-up of new methods are also activities of the core facility. The core facility promotes and fulfils the 3R’s (reduction, replacement, refinement) in animal experiments.

The European Mouse Mutant Archive EMMA is a repository for the collection, archiving (via cryopreservation) and distribution of relevant mouse mutant strains essential for basic biomedical research. EMMA is organized as a decentralized archive of 14 partners in 11 European countries. The partners have established common operating procedures and highest-quality standards. The Biocenter Oulu transgenic core facility serves as the Finnish EMMA node. Since 2013, EMMA has been part of the Infrafrontier research infrastructure (RI). (www.infrafrontier.eu). Infrafrontier supplies GM mouse models to researchers economically and efficiently (archiving and distribution), and provides information about the phenotypic features of mouse models (systemic phenotyping), such as analysis of development, morphology, physiology, metabolism and behaviour. The Infrafrontier Technical Working Groups meet twice a year to discuss and plan development of new methods and improvements of the ones in use. The Infrafrontier RI closely collaborates with and contributes to the International Mouse Phenotyping Consortium IMPC (www.mousephenotype.org). The IPAD-MD project (Research Infrastructure for Phenotyping, Archiving and Distribution of Mouse Disease Models) addresses global cooperation and coordination between Infrafrontier and complementary research infrastructures worldwide, contributing to the global effort of the IMPC.

Funding to provide repository services, cost-free cryopreservation of germ cells and embryos, generation of GM mouse strains from embryonic stem (ES) cells, and re-derivation and distribution of mouse strains is provided by the EC Infrafrontier-I3 grant for 2013–2016. The Finnish unit is archiving 70 mouse strains in 2013–2016, largely from the Finnish research community, providing cost-free generation of six mouse strains from ES cells as a transnational access project, and distributing mice according to orders.

Finnish participation in Infrafrontier was included in the Finnish Research Infrastructure Roadmap in 2009 and 2013, and the University of Oulu is representing Finland as a partner in the legal entity, Infrafrontier GmbH.

Recent Progress

We have continued to provide up-to-date services concerning GM mouse generation, re-derivation and archiving to customers in Oulu, as well as other universities in Finland and abroad. Since 2000, more than 200 gene-targeted mouse strains have been generated, in addition to a large number of transgenic mouse strains. In 2015 we served 57 customers involved in a total of 254 projects. As a special project, cleaning and re-derivation of 40 mouse strains at the newly disinfected animal facility SPF barrier unit was performed in 2015, and cleaning of 17 strains will be finished in 2016.

Sperm cryopreservation and in vitro fertilization (IVF) were started as new services in 2010, and currently mouse strains are cryopreserved as sperm and two-cell embryos generated via IVF. In 2015, a total of 27 mouse lines were cryopreserved for in-house use, and 22 strains archived in EMMA. Generation of iPS cells (induced pluripotent stem cells) and production of mice from these cells have been accomplished. However, the more recently set up 2i method for efficient isolation of ES cells from mouse embryos is the method of choice in most cases. Using this method, we have already generated more than 100 ES cell lines in seven projects. These cells will mainly be used for in vitro studies, but we have also performed a second round of gene targeting and generated a mutant mouse strain using ES cells generated by this method.

The BCO Transgenic core facility serves as the Finnish Infrafrontier-EMMA node, a national archiving facility. Full national and international services in archiving and distribution of mouse lines have been provided in Oulu since the start of the operational phase of the Infrafrontier-I3 project in 2013. So far, a total of 37 mouse strains originating in Finland have been archived in EMMA, and an additional 16 strains were submitted to the EMMA archive to be archived in 2016. Furthermore, six gene-targeted mouse strains have been generated as an Infrafrontier transnational access service.

Since July 2011 the BCO Transgenic core facility has served as a distribution node of the Wellcome Trust Sanger Institute, located in Hinxton, Cambridgeshire (UK), which generates new mutant mouse strains as part of the EUCOMM (European Conditional Mouse Mutagenesis) project. In 2015, 16 mouse strains were shipped to customers in Australia, China, Japan, the U.K. and the U.S.A. as live mice or embryos.

Research
Currently our main focus is in establishment of new methods in generation and cryopreservation of genetically modified mice. Therefore, technological development related to gene modification in mice belongs to our research activities. The latest addition is generation of gene-edited mice via CRISPR/Cas9 technology. A method for site-specific modification of the genome in early mouse embryos using cell-permeable Cre fusion protein was also set up, and we have also successfully tested and added ovary transplantation to our methods repertoire, and started using laser-assisted morula injection for generation of chimeric mice, reducing the number of donor mice. Genotyping of blastocyst-stage embryos was established as part of quality control of EMMA services, and tests including shipment of embryos and sperm economically and efficiently without liquid nitrogen were performed. Most recently, a method for cryopreservation of oocytes via vitrification was successfully set up.

We are also studying the functions of heparan sulfate proteoglycans (HSPGs), which influence biological processes by interacting with important signalling molecules. They also bind water, and several extracellular matrix components have affinity to heparan sulphate (HS). Perlecan is a large HSPG found in all basement membranes (BMs) and also in the tissue stroma of liver and cartilage, where no BMs are present. It carries three or four HS side chains that have been shown to bind growth factors. One of our goals is to elucidate the functions of HS chains in BMs, perlecan being the primary model proteoglycan, and study if variations in HS content could have a role in symptoms of BM-connected diseases. For this, we have generated mice in which the perlecan gene is targeted so that the attachment sites of HS side chains are deleted.

Future Goals

The transgenic core facility will continue to provide high-level services to researchers and keep updated in new technology, especially as regards novel genome-editing tools.

We will be actively involved in national and international activities related to GM mice. In the Infrafrontier-I3 project, the goal is cryopreservation of 70 mutant mouse lines in Finland by the end of 2016.

Publications 2015-

Aro E, Salo AM, Khatri R, Finnilä M, Miinalainen I, Sormunen R, Pakkanen O,Holster T, Soininen R, Prein C, Clausen-Schaumann H, Aszódi A, Tuukkanen J, Kivirikko KI, Schipani E, Myllyharju J. Severe extracellular matrix abnormalitiesand chondrodysplasia in mice lacking collagen prolyl 4-hydroxylase isoenzyme II in combination with a reduced amount of isoenzyme I. J Biol Chem 290(27):16964-78, 2015.

Colombelli C, Palmisano M, Eshed-Eisenbach Y, Zambroni D, Pavoni E, Ferri C, Saccucci S, Nicole S, Soininen R, McKee KK, Yurchenco PD, Peles E, Wrabetz L, Feltri ML. Perlecan is recruited by dystroglycan to nodes of Ranvier and binds the clustering molecule gliomedin. J Cell Biol 208(3):313-29, 2015.

Hurskainen T, Kokkonen N, Sormunen R, Jackow J, Löffek S, Soininen R, Franzke CW, Bruckner-Tuderman L, Tasanen K. Deletion of the major bullous pemphigoid epitope region of collagen XVII induces blistering, autoimmunization, and itching in mice. J Invest Dermatol 135(5):1303-10, 2015.

INFRAFRONTIER Consortium. INFRAFRONTIER--providing mutant mouse resources as research tools for the international scientific community. Nucleic Acids Res 43 (Database issue):D1171-5. 20, 2015.

Ronkainen J, Huusko TJ, Soininen R, Mondini E, Cinti F, Mäkelä KA, Kovalainen M, Herzig KH, Järvelin MR, Sebert S, Savolainen MJ, Salonurmi T. Fat mass- and obesity-associated gene Fto affects the dietary response in mouse white adipose tissue. Sci Rep 18, 5:9233, 2015.

Scavizzi F, Ryder E, Newman S, Raspa M, Gleeson D, Wardle-Jones H, Montoliu L, Fernandez A, Dessain ML, Larrigaldie V, Khorshidi Z, Vuolteenaho R, Soininen R, André P, Jacquot S, Hong Y, de Angelis MH, Ramirez-Solis R, Doe B. Blastocyst genotyping for quality control of mouse mutant archives: an ethical and economical approach. Transgenic Res 24(5):921-927, 2015.

Research Group Members


Project Leader:
Raija Soininen, Docent, Ph.D. (University of Oulu, Academy of Finland)

Researcher:
Reetta Vuolteenaho, Ph.D. (Biocenter Oulu, UO strategic funding targeted for BF operations)

Laboratory supervisor:
Jonna Ranta M.Sc. (University of Oulu)

Technicians: 3,5 (University of Oulu and Biocenter Oulu, UO strategic funding targeted for BF operations, Infrafrontier-I3)

National and International Activities

Biocenter Finland Model Organisms network FinnMouse (chair)

NordForsk project

Researcher network project Nordic Infrastructure for Mouse Models NorIMM (coordination) 2014-2016

EU project

EC FP7 project Infrafrontier-I3 (partner), 2013-2016

ESFRI project

University of Oulu is a founding member in Infrafrontier GmbH, which was created to coordinate the transnational acitivities of the Infrafrontier RI. Other founding members are the Helmholtz Zentrum München, Germany, the Centre National de la Recherche Scientifique, France, the Institute of Molecular Genetics of the Czech Academy of Sciences, Czech Republic, and the Biological Research Center 'Alexander Fleming', Greece.

Last updated: 10.5.2017