Purpose
The gene targeting facility specialises in the production of transgenic mouse models. We use CRISPR/Cas9 to target the genome at the zygotic stage. This bypasses the need for targeting mES cells first, reducing the process of genetically modified mouse production by months. We can generate a wide variety of mouse models using this method including:
Point Mutations
The estimated number of nucleotide differences between two human genomes is estimated to be ~ 5 million bp and are called Single nucleotide polymorphisms (SNP). These differences can give rise to differences in protein function and can also impact gene expression. Mouse models are the perfect model to recapitulate these SNP. In protein coding regions, SNP can change the amino acid sequence, potentially resulting in an inactive protein. SNP found in non-coding regions can be found in regulatory elements and in DNA motifs, thus point mutation in mouse models allows users to investigate their role in vivo.
Knock-out mice, conditional and deletions
CRISPR/Cas9 can be used to induce double stranded breaks (DSB) near a PAM sequence (NGG) in the genome. These breaks are repaired by microhomology end joining (MMEJ) or non-homology end joining (NHEJ), resulting in indels. Indels in protein coding regions lead to frameshifts if they are not divisible by 3, resulting in a premature stop codon. In cases where zygotic KO are not viable, we can create conditional mouse lines whereby a critical exon is flanked by loxP sites. The floxed mouse can then be crossed with a mouse carrying CRE recombinase under control of a tissue specific promoter. We can also generate mouse models with deletions with varying lengths being assessed on a case by case basis.
Gene Tagging
In addition to changing single nucleotides, we can also add additional sequence into genes. Most commonly onto the 5’ or 3’ of the gene, enabling tagging of the endogenous gene. Various peptide tags can be appended onto the gene of interest such as FLAG-, V5-, Myc- and His-tags are just a few examples possible. Larger tags such as GFP and mAID are also possible but are dependent on sgRNA sites with high activity. Ideally, we would ideally target near the stop codon (3’UTR) of the gene to avoid indels near the ATG site. Tagging allows visual tracking of the endogenous gene, protein expression when validated antibodies are not available or even clearance of the tagged protein.
Guidelines
The Gene Targeting Facility is currently only available to users affiliated with the º£½ÇÉçÇøÂÛ̳.
Please contact Asif Nakhuda (asif.nakhuda@babraham.ac.uk) in the first instance.