CNR-IBC/IBCN Monterotondo’s mutant strain production unit (MMP), from 2008 partecipated in EUCOMM (European Conditional Mouse Mutagenesis) project, that is a cornerstone of the International Knockout Mouse Consortium (IKMC). Under the prof. Glauco P. Tocchini-Valentini scientific supervision, they produced about 106 mutant mouse lines from that date, by ESC blastocyst injection, feeding of mutant mice, the large scale mouse phenotyping projects EUMODIC (European Mouse Desease Clinic), and IMPC (International Mouse Phenotype Consortium). They was also member of EUCOMM Tools project in which they produced as requested from their work package up to 50 (53) mouse mutants CRE drivers line, as well as dissection from these lines to detect the effective tissue specific CRE expression of the mutants produced. These line are already crypreserved in EMMA and ready for distribution, to the scientific community. The actual production capability of this team is about 20-35 mutant lines per years. Thanks to the involvement in IMPC CRISPR/CAS9 discussion and pilot, they actually obtain this innovative technology, that is now part of the active mutant mouse production.
1: de Angelis MH, et al. Nat Genet. 2015 Sep;47(9):969-78.
Analysis of mammalian gene function through broad-based phenotypic screens across a consortium of mouse clinics. doi:10.1038/ng.3360. Epub 2015 Jul 27. PubMed PMID: 26214591; PubMed Central PMCID:PMC4564951.
2: Bradley A, et.al, Mamm Genome. 2012 Oct;23(9-10):580-6.
The mammalian gene function resource: the International Knockout Mouse Consortium. doi: 10.1007/s00335-012-9422-2. Epub 2012 Sep 12. PubMed PMID: 22968824; PubMed Central PMCID: PMC3463800.
Animal Models and Gene Therapy Core
The Transgenic Animals Facility is active within CNR since 1999 and focuses on the generation of genetically modified mice (transgenic, knockout, knockin).The facility applies all the relevant standard transgenic technologies but also focuses on the latest methods to introduce genetic modifications in vivo: Zinc Finger nucleases (ZFN), Transcription activator-like effector nucleases (TALENs), Clustered regulatory interspaced short palindromic repeat (CRISPR/Cas-based RNA-guided DNA endonucleases). The service is offered oa collaborative and/or contributive basis also providing support and advice on projects and strategies involving the use and/or the generation of animal models.
The services of the facility include:
- purification of transgenic DNA constructs;
- creation of transgenic mouse models by pronuclear microinjection of the DNA constructs in the embryos at the zygote stage and realization of knockout (KO) or knockin (KI) models by injection of mutagenized embryonic stem cells into blastocysts;
- microsurgery (vasectomies, uterine embryo transfer);
- collection, fixation and staining of murine embryos at various stages of development;
- isolation and culture of embryonic stem cells (ES) and primary fibroblasts from embryos;
- genotyping of mutant mice by PCR;
- management of mutant mice colonies (breeding, weaning, backcrossing, rederivation);
- cryopreservation of sperm and two-cells embryos, in vitro fertilization;
- collection of tissues and blood for biochemical, histological, and serological analysis;
- hormone treatments, pharmacological, and gene therapy on mice.
The Service organized a theoretical/practical course within the CNR Training Programs : Corso ‘Metodologie di riderivazione di linee murine’ and participates as teaching staff to lectures and practice on genetically modified animals in the annual course: ‘Science of Laboratory Animals’ (Recognition FELASA, Federation of European Laboratory Animal Science Associations) Cat B (No: 023 / 09).