RMCE for Large Fragment Knock-in Mouse Models

Site-Specific Recombination & Related Genome Modification Strategies

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.

Examples of SSR-based strategies:

• Cre-lox system (a.k.a. gene floxing) - temporal and spatial control of gene expression
• Recombinase-mediated cassette exchange (RMCE) – insertion of large gene sequences (up to 300 kb); generation of several mutant models for comparative studies

Applications of Recombinase-Mediated Cassette Exchange (RMCE)

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.

Large Fragment Knock-in Models – Humanization, Transgenic, Reporter, Conditional

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!

Developing Mutant Models for Comparative Analysis

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.

Large Fragment Knock-in Mouse Models

Cyagen has successfully generated large fragment knock-in mouse models using TurboKnockout^® or Targeted Gene Editing, 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.