This project has received funding from the European Union’s Horizon Europe research and innovation programme under grant agreement No 101131669.
INFRAFRONTIER ERIC, along with its partners Mary Lyon Centre at MRC Harwell, University of Oulu and PHENOMIN-ICS, is pleased to announce the launch of a servcie aimed at the generation of mutant embryonic fibroblasts and mouse embryonic stem cells.
This INFRAPLUS service aims to support scientists in developing mutant cell models that, when scientifically appropriate, serve as alternative to animal models for relevant experimentation. It promotes the 3Rs principles (Replacement, Reduction, and Refinement) by minimising animal use whenever relevant cell models can be employed instead. (see INFRAFRONTIER’s stance and contribution to 3Rs and animal welfare HERE).
Two services are offered:
Mouse Embryonic Fibroblasts (MEFs) Isolation
The aim of this service is to provide MEFs isolated from existing mouse lines.
The service includes:
- If needed, revitalisation of the mouse lines.
- If needed, one mating step to obtain the required zygosity for the cell model relevant to each mutation.
- If needed, mating to generate embryos for MEFs isolation:
a. Embryos at E13.5 to E16.5 will be used to establish independent fibroblast cultures from each individual embryo.
b. A maximum of 4 successful matings will be performed. - Isolation of MEFs from embryos:
a. The number of embryos processed will depend on genotype frequency and potential sub-viability.
b. A biopsy will be collected from each embryo to determine genotype and gender. Sufficient information, such as the sequence of the targeted allele, must be provided in advance in order to derive a suitable genotyping strategy. Sufficient information is required in advance to design the genotyping strategy.
c. Each embryo-derived culture will be expanded to confluence in a 9- or 14-cm dish (or equivalent) and cryopreserved in three to four aliquots. - Frozen aliquots, along with genotype, will be delivered to the service user:
a. One aliquot will be retained until the user confirms receipt of the cell line.
b. MEF lines will be provided with the objective of including both sexes, when biologically feasible.
MEFs carrying multiple mutations at different loci and/or expressing various transgenes are within the scope of this service; however, the PI should note that a maximum of two breeding steps, done at the INFRAFRONTIER center, is included (additional steps will need to be covered by the PI).
Mouse Embryonic Stem Cells (mESC) Derivation
The aim of this service is to provide mutant mESCs derived from existing mouse lines.
The service includes:
- If needed, revitalisation of the mouse lines.
- If needed, one mating step to obtain the required zygosity for the cell model relevant to each mutation.
- If needed, mating to generate blastocysts for mESC derivation:
a. Blastocyst-stage mouse embryos will be used to establish independent stem cell cultures from each individual embryo.
b. A maximum of 4 successful matings will be performed. - Derivation of mESC from blastocysts:
a. The number of embryos processed will depend on genotype frequency and potential sub-viability.
b. An aliquot of cells will be taken once each line growth is established to determine their genotype and gender.
c. Cultures from each embryo will be amplified to a confluent 5 cm dish or one well of a multiwell plate or equivalent and frozen in three or four aliquots. - Additional molecular validation (e.g., mRNA expression analysis, chromosome counting, or MiniMUGA genotyping array) may be performed at the discretion of the providing institute, following consultation with the PI and depending on the intended use of the cells.
- Frozen aliquots, along with genotype, will be delivered to the service user:
a. One aliquot will be retained until the user confirms receipt of the cell line.
b. mESC lines will be provided with the objective of including both sexes, when biologically feasible.
mESCs carrying multiple mutations at different loci and/or expressing various transgenes are within the scope of this service; however, the PI should note that a maximum of two breeding steps, done at the INFRAFRONTIER center, is included (additional steps will need to be covered by the PI).
Cell lines will be derived using our optimized protocol, which generally produces high-quality cells with preserved differentiation potential; however, the pluripotency status of the resulting cell lines cannot be guaranteed.
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