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.
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.
Gene knockin mice are indispensable for biomedical research, such as the discovery of new drug targets and preclinical pharmacodynamic evaluation.
Knock-in mouse models can be constructed by CRISPR/Cas9 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.
TurboKnockout® Gene Targeting | CRISPR/Cas9 Gene Editing | |
---|---|---|
Approach | Homologous recombination in ESC by our proprietary TurboKnockout® technology | CRISPR/Cas9 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
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