Core Facility for Gene Transfer and Mutagenesis
Genetically modified mice and cell lines are fundamental tools in biomedicine. In the past few years incredible progress was made in the available technologies, allowing a leap in precision, timing and costs. The scope of the Core Facility for Gene Transfer and Mutagenesis (CFGTM) is that of generating both genetically modified mice using either CRISPR Cas9 or ES cells as appropriate, and CRISPR Cas9-KO cell lines and provide them as a service to the scientific community.
The CFGTM can count, within the Mouse Facility of the Molecular Biotechnology Center, on a dedicated mouse room equipped with filtered cages and hosting SPF mice for generating donor and recipient mice for the experiments. Dedicated ES cells Tissue Culture Facilities allow the efficient generation of targeted ES cells. Two microinjection apparatuses allow the microinjection of both one-stage embryos and blastocysts. Electroporation of one-stage embryos with recombinant Cas9 and guide RNAs is also possible.
The facility offers the production of genetically modified mice and cell lines for both internal and external users.
We recently introduced the CRISPR/Cas9 system to generate KO and Kin mice, which allows unprecedented efficiency (3-4 months as opposed to 1-1,5 years with the traditional ES cell-based gene targeting approach). Moreover, with this system multiple genes can be simultaneously targeted. All together, these features allow to save about one third of the cost with respect to ES cells-based approaches.
C57BL/6 and FVB in-house bred mouse strains are used.
The service includes strategy planning including screening strategies, construct design, construct generation, DNA purification and microinjection into the male pronucleus of 150 fertilized oocytes. Embryos are transferred into foster mothers and pregnancies are monitored. The investigators then receive tail biopsies from 14 days old mice for genotyping. On demand, screening can also be provided by the Facility. Positive TG mice will be shipped at the user’s expenses, at 4 weeks of age.
CRISPR/Cas9 has rapidly become the most widely used approach to perform gene editing in mice and cells. It is based on a guide RNA that is designed against the region to target and contains a sequence that is recognized and bound by the Cas9 protein. This RNA guides Cas9 to the target DNA, where the enzyme introduces a double strand break at a precise region. The break is then repaired via either the Non Homologous End Joining (NHEJ) pathway or the Homology Directed Repair (HDR) pathway, in the presence of a donor DNA with homologous sequences. NHEJ is used to generate KO models, HDR for K/in models.
We directly electroporate into mouse zygotes recombinant Cas9 and guide RNA to generate KO mice, and incude donor DNA to generate K/in mice. This strategy allows to obtain gene-edited mice bypassing the need to manipulate ES cells, dramatically reducing the time required (three to four months against 1-1.5 years), and the costs involved in ES cells culture.
EP Embryos are then surgically transferred into pseudopregnant foster mothers, pregnancies are monitored, and born pups undergo tail biopsy at 3 weeks of age. Tail biopsies are genotyped via PCR amplification of the predicted edited region followed by sequencing. Targeted mice are finally shipped to the customer.
Three different models are generated:
- KO mice via introduction of indel mutations or specific deletions.
- K/in models via electroporation of a guide RNA and a single strand donor oligo carrying the mutation of interest.
- Further alternatives such as targeted nucleotide substitutions (C to T or G to A) or conditional gene targeting via loxP sites insertion can be implemented.
Traditional gene targeting in mouse embryonal stem cells (ES cells) may still be convenient for the generation of conditionally mutant alleles, by the insertion of LoxP or Flip recombinase sites into introns and the generation of so-called floxed or flipped alleles, fully functional but ameanable to Cre or Flp recombinase-mediated deletion. Coupled to crosses with suitable recombinase-expressing transgenic mice or to infection with recombinase-expressing viral vectors, tissue-specific or inducible gene inactivation can be achieved. These approaches require long regions of homology that so far have not been efficiently used in the Cas9 system.
The construct needs to be carefully designed, in parallel with the screening strategy to distinguish between wild type and mutant alleles. Suitable probes for genomic DNA need to be designed and experimentally tested.
The homology regions are excised by recombination from a BAC genomic clone, and combined with LoxP or Flp sites and a floxed or flipped-flanked Neo resistance cassette.
In case the customer already has a vector, we will need to control its suitability to our conditions.
Even though it is rare that no recombinant clones are recovered, we cannot guarantee the generation of homologous recombinant clones, since some genomic locations appear to be resistant to homologous recombination.
The linearized vector will be electroporated into ES cells, which will then be subjected to the appropriate selection procedure. Antibiotic resistant clones will be picked (minimum 200), expanded and screened by genomic Southern blot.
ES cell lines derived from chinchilla 129 strains will be injected into blastocysts from C57/Bl6xBalb/C F1 hybrids, while C57/Bl6 ES cell lines into Balb/C embryos. A minimun of 50 or 100 embryos will be transferred, depending on the conditions. The injection of customer-provided ES cells can also be performed.
The Facility can generate suitable lentiviral vectors for both purposes, including: cloning of the sequences for the expression of the guide RNA or the shRNA, restriction control and sequencing, generation of the virus via infection of 293T cells, infection of the customer-provided cells. Customer-provided plasmids or viruses can also be used.
CRISPR/Cas9-mediated KO cell lines will be generated via infection with lentiviral vectors expressing Cas9 and the suitably designed guide RNA, followed by selection and genetic screening. We can also generate the vectors. Results and costs will depend on the cell line, please enquire.
shRNA-mediated stable KD will be obtained via infection with lentiviral vectors expressing suitable shRNAs, followed by selection. Screening by RT-PCR can be either provided as a service or performed by the investigator.
Any kind of cloning can be performed by either traditional approaches or recombination in bacteria. We have a battery of the most common cloning plasmids. In case uncommon vectors are needed, these must be provided by the customer. The service includes designing the cloning strategy, performing the cloning, verification of the correct clones by restriction analysis and sequencing if needed. At request maxi- or midi-prep scale DNA amounts can be provided.
Other services, such as production and purification of recombinant proteins, generation and purification of monoclonal antibodies, and generation and production of recombinant AAV vectors, can also be provided.
All prices are on demand.