Knock-in (KI) up to 300 kb | Conditional Knockout (cKO) up to 100 kb
Since the beginnings of gene editing research, the ability to accurately modify large genomic regions has remained a primary driver of innovation in the field. Although gene editing technologies have been continuously refined, the size of the modifiable region is still constrained depending on the methods used.
Developing mouse models with large genomic modifications has proven to be a challenging task for many institutional cores and companies. Research of whole gene replacements - used in gene replacement therapy – often pushes the limits of gene editing technologies. Large fragment knock-ins (LFKIs) – similarly limited - are used to develop conditional, reporter, transgenic, and humanized mouse models. For whole gene replacement and large fragment knock-in projects, the recombinase-mediated cassette exchange (RMCE) approach – a type of site-specific recombination - can provide such mouse models with knock-ins up to 300 kb.
Genetic recombination using site-specific recombinases (SSRs) exchanges DNA between segments which share a certain degree of sequence homology, providing a targeted way to introduce the gene of interest. SSRs rearrange DNA within recombination sites that typically range from 30-200 nucleotides in length. Recombination occurs upon the SSRs’ binding of the recombination site - consisting of two motifs with a partial inverted-repeat symmetry. The relative orientation and molecular location of these sites dictates the type of recombination event that occurs, whether genes are inverted, excised, or inserted. Notably, an insertion requires intermolecular recombination (sites situated on two different DNA molecules) with one DNA molecule that is circular.
RMCE is often recommended for conditional, reporter, humanized, and transgenic mouse model projects due to its ability to efficiently insert of large gene fragments.
Recombinase-mediated cassette exchange (RMCE) implements site-specific recombination to achieve systematic, repeated modification of higher eukaryotic genomes. The RMCE approach is suitable for whole gene replacement and large fragment knock-in mouse model projects; it is often used to develop conditional, reporter, transgenic, and humanized mouse models.
RMCE technology integrates the gene of interest (GOI) by exchanging a preexisting gene cassette (gene and recombination site) with an analogous cassette containing the GOI. To achieve this, the wildtype gene is flanked by two unique recognition sites and the DNA plasmid containing the mutated GOI is flanked by the same two recognition sites – a recombinase (e.g. Cre) is used swap the wildtype gene for the mutated GOI.
The recombinase-mediated cassette exchange (RMCE) approach remains a powerful tool for the insertion of large gene fragments, and may be used to efficiently generate conditional, reporter, and transgenic mouse models. RMCE provides highly efficient transfer of genes of interest (GOIs) and is readily adapted to generate humanized mouse models – reproducing the human expression patterns in mice. With our project strategies, we have used RMCE-based techniques (with BAC fusion and 3 targeting rounds) to achieve large-fragment knockin (LFKI) humanization and genetic modifications for regions up to 300 kb!
Additionally, recombinase-mediated cassette exchange (RMCE) technology may be used to generate several mutant models for large-scale mutation screens, characterization of domain-specific protein functions, and other comparative studies. RMCE enables insertion of a mutation directly into a parental embryonic stem (ES) cell clone, which is engineered for use alongside different targeting vectors to readily generate additional mutant cell and mouse lines. Given that only one parental ES clonal line is required to generate a full range of unique mutant cell lines, and subsequent mutant mouse lines, RMCE can save time and reduce project costs. RMCE enables researchers to generate point mutations, large gene mutations, and even replace a wildtype gene with a different GOI in a highly efficient and cost-effective manner for large-scale genomic studies.
Cyagen has successfully generated large fragment knock-in mouse models using TurboKnockout® or CRISPR, across both endogenous gene loci and Rosa26 loci. We provide a complete range of services, from generation of the engineered parental ES cell line through delivery of research-ready custom mouse models.
Using data from thousands of knock-in mouse model projects completed by our team, we have collected new information demonstrating how our gene editing technologies push the boundaries of modifying large genomic regions. Below, we have outlined Cyagen’s large fragment knock-in (LFKI) capabilities across our gene editing technologies.
|TurboKnockout®||CRISPR/Cas9 Gene Editing|
|Approach||Homologous recombination in ESC by our proprietary TurboKnockout® technology||CRISPR/Cas9 nuclease mediated gene targeting by pronuclear injection|
Large fragment knockin
Large fragment knockin
|Knock-in (KI) fragment size limits||~20 kb per round of gene targeting
RMCE Humanization: ~ 300 kb
|Endogenous: ~15 kb
Rosa26/H11: ~12 kb
|Conditional Knockout (cKO)||Single target: ~7 kb
Double target: ~100 kb
|Donor vector: ~7 kb
ssDNA: ~100 kb
|Donor backgrounds||Mouse strains: C57BL/6, BALB/c||Mouse strains: C57BL/6, FVB
Rat strains: Sprague-Dawley (SD), Long Evans
|Turnaround time||6-8 months||5-7 months|
From strategy design through to delivery of research-ready custom mouse models, Cyagen offers complete outsourcing for all your animal model needs. Cyagen’s gene editing services are unparalleled in efficiency of developing rodent models with a guaranteed genotype. We even offer price matching to help ensure researchers get the best deal for their study.
Contact us with your gene target(s) and our custom model generation experts will be in touch with you.