Gene knock-in (KI), a.k.a. knockin, mice are generated by introducing specific mutations or exogenous genes into specific sites of the target gene through homologous recombination, so that the expression of the gene knockin may be tracked through the expression of a reporter.
What Is a Gene Knock-in?
A gene knock-in (KI) refers to the introduction of a specific gene sequence into the target gene locus, such as point mutations (simulating human genetic disease), or reporter genes (such as EGFP, RFP, mCherry, YFP, lacZ, luciferase, etc.) or cDNA (expressed for functional genomics) to specific sites of target gene by homologous recombination, so that the expression of reporter gene or other cDNA was consistent with that of target gene. By definition, if the reporter gene or cDNA is used to replace the mouse gene, knock-out and knock-in will occur simultaneously. The humanized mouse model can also be obtained by replacing the endogenous mouse gene with human gene or inserting human cDNA into the ATG position of the endogenous mouse gene.
What Are Knockin Mice Used For？
Gene knockin mice are indispensable for biomedical research, such as the discovery of new drug targets and preclinical pharmacodynamic evaluation.
- ● Imitating human genetic mechanisms
- ● Exploring human pathogenic mechanisms
- ● Humanized mouse generation, to accelerate drug research and development
- ● Tracing gene expression
- ● Lineage tracing to trace the origin of cells
- ● And more
Types of Gene Knock-in Mouse Models
- ● Regular gene knock-in: Insert the foreign gene sequence into the designated site.
- ● Rosa26 knock-in: Introduce the foreign gene at the Rosa26 locus - a "safe harbor" locus - so exogenous gene site insertion will not affect the expression of other genes.
- ● Point mutation: Introduce the point mutation to the corresponding position of mouse homologous gene; often used to simulate such human genetic diseases.
- ● Conditional point mutation: Combining point mutation with Cre/loxP system to realize tissue-specific and/or temporally controlled point mutation. Conditional models circumvent problems with mutations resulting in embryonic lethality.
- ● Humanization: The endogenous mouse gene is replaced with the homologous human gene to construct humanized mouse models of diseases, which are used in research across immunity, physiology, antibody drug screening, efficacy evaluation and more.
What Methods are Used to Construct Gene Knock-In Mouse Model?
Knock-in mouse models can be constructed by CRISPR-Pro or embryonic stem (ES) cell-mediated gene targeting technology. With our proprietary TurboKnockout® Gene Targeting services, Cyagen can generate large fragment knockin (LFKI) mice with fragment sizes up to 300 kb.
Cyagen Knock-In Mouse Capabilities
| TurboKnockout® Gene Targeting | CRISPR-Pro Gene Editing | |
|---|---|---|
| Approach | Homologous recombination in ESC by our proprietary TurboKnockout® technology | CRISPR-Pro nuclease mediated gene targeting by pronuclear injection |
| Applications | Conditional knockout Point mutation Large fragment knockin Humanization |
Conditional knockout Point Mutation Large fragment knockin Global knockout |
| 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 |
Other Services Cyagen Offers for Knockin Mice
