The mission of the FACS Core is to provide access to high quality flow cytometry, fluorescence-activated cell sorting (FACS) and mass cytometry (CyToF). The Core aims to provide highly trained staff and the most modern equipment to all prospective users, in order to facilitate elite research through the use of flow cytometry.
The Core has four modern sorters, a BDFACS Aria IIu, BDFACS Aria III, BD Influx and Sony MA900 and five cytometers, a ThermoFisher Attune with an Auto Sampler, a Beckman Coulter Gallios, two BD Canto II’s and a BD Symphony. Users also have access to an imaging cytometer, the Amnis ImageStream MKII. There is also a Meso QuickPlex SQ120 Multiplex electrochemiluminescence imager available for booking. Finally, the Helios, a mass cytometer (CyToF) is available
Genome Engineering Platform
In close collaboration with the VIB microinjection unit, we provide all KUL laboratories with access to research tools in genome engineering from design to cell lines or mice, respectively.
We can assist you at every step of your CrispR/Cas9 project. We offer consultations as well as technical assistance. The all-inclusive service for a genome editing project includes:
- The identification of functional guide RNA
- Suggestions for the experimental design for knockouts, deletions, insertions/knockins, overexpression and conditional alleles
- Cloning support and set-up of a screening strategy
- use of mouse ES cells (JM8.A3 line) if necessary
- in case of cell lines we can provide cell sorting for enrichment or clonality
You can choose to perform individual steps yourself or choose for the all-inclusive service. For differences in pricing please consult our website or contact u for details.
Genome Engineering Services
CrispR/Cas has emerged as a powerful tool for genome editing. The genome editing platform uses CrispR/Cas to permanently mutate a gene of interest. CrispR guide RNAs guide a DNA endonuclease (SpCas9, SpCasN, SaCas9 or Cpf1) to the gene locus of interest where a double-strand break is generated. In the absence of a donor DNA this double-strand break will be repaired by non-homologous end-joining resulting in indels and thus a knock-out. In the presence of a donor DNA, repair can take place by homologous recombination (knock-in).
We offer the use of four Cas enzymes to manipulate cell lines of interest as well as mouse embryonic stem cells if downstream mouse generation is desired.
KO Mouse generation
Currently, the most common application of CrispR/Cas is the generation of knock-out alleles. The preference of the cells to repair double-strand breaks introduced by the Cas9 endonuclease is non-homologous end-joining (NHEJ). This imperfect repair mechanism results in insertions or deletions of nucleotides which, if in an exon, can result in a shift of the open reading frame. Consequently, a premature stop codon will appear downstream of the mutation and result in a truncated transcript.
You can bring your own tested crisprs or we can help you find appropriate crisprs.
For knock-outs in cell lines we offer the possibility to bulk sort your cells to enrich after transfection and to single-cell sort to generate individual clones.
To generate knock-out mice, complexes of crispr RNA and Cas9 protein – so-called ribonucleoproteins – are injected into early embryos.
KI Mouse generation
Some studies may require the modification of a gene by exchange or addition of sequence. Examples are
- The insertion of point mutations that resemble human disease-causing polymorphisms
- Tagging of a protein of interest with an epitope, such as Flag or hemagglutinin.
- The gene of interest can be turned into a conditional allele to allow temporal, spatial or cell type-specific knock-outs.
- The gene of interest can be linked to a fluorescent reporter (such as GFP) or a recombinase (such as Cre). In the case of GFP, active transcription is visualized. In the case of Cre, live and past activity of the gene can be followed.
In addition to a functional gRNA knock-in experiments require a donor DNA that serves as a template for homologous recombination. Our team can help you with the design as well as the cloning of your donor DNA.
Complex knock-ins will be performed in mouse embryonic stem cells. Those cells will subsequently be injected in to blastocysts to generate chimeric mice. For smaller knock-ins, such as epitope tags or point mutations, we try to to achieve knock-ins by direct pronuclear injection.