The term “knockin mouse” refers to a mouse strain with targeted insertion of a mutation, transgene or other DNA sequence at a specific site in the mouse genome. The inserted sequence can replace part of the mouse genome (e.g. replace a wild-type mouse sequence with a mutant version or replace a whole mouse gene with a human gene) or it could be a point insertion where exogenous DNA such as a transgene is inserted at a desired location without deleting any mouse sequence.

Most kinds of knockin mice can be produced in approximately 6-9 months, with confirmed heterozygous animals as the deliverable. More complex alleles such as whole gene replacements can be produced in approximately 9-12 months.

Our default strain is C57BL/6J, but we can produce knockin alleles in a variety of other strains, depending on customer needs.

Yes. Most human genes have a mouse counterpart and gene function is usually at least partially conserved between species. Important protein functional domains are often highly conserved between human and mouse. By careful analysis we can identify the corresponding part of the mouse gene to generate a mutation mimicking the human disease mutation.

In most cases, yes! If there is at least one active CRISPR guide RNA overlapping the mutation site, the mutation can generally be inserted without any extraneous mutations such as adjacent “silent” wobble codon mutations. When possible, we try to avoid inserting any unnecessary changes that might inadvertently alter the phenotype of the animals through changes in RNA folding or translational efficiency.

Protein tags are usually attached to the N- or C-terminus of proteins to allow their tracking or analysis in endogenous mouse tissues. The generation of a tag mouse model involves identifying CRISPR guide RNAs that cut at the appropriate location in the mouse gene (i.e. near the ATG start codon or the stop codon). A donor DNA is generated to work with the CRISPR reagents to promote seamless insertion of the desired tag immediately downstream of the ATG start codon or upstream of the stop codon.

Reporter genes encode proteins that allow detection through fluorescence (e.g. green fluorescent protein), bioluminescence (e.g. luciferase) or other enzymatic activity (e.g. lacZ). Reporter genes can be targeted in the mouse genome to tag or co-express with an endogenous mouse gene. This allows analysis (“reporting”) of the expression pattern and expression level of the endogenous gene. Reporter gene mice can be used to label specific cell types where the target gene is expressed, measure changes in endogenous gene expression triggered by biological signals, or mark protein interaction partners for identification of protein complexes and networks.

For knockin by direct CRISPR injection in mouse embryos, the upper size limit for successful knockin is probably around 15-20 kilobases. However, much larger insertions can be achieved in embryonic stem cells using CRISPR with selectable markers in the donor vector. In that setting the upper size limit for knockin insertions is likely dictated by the ability to produce a suitable donor with the desired sequence.
We have successfully achieved knockin of sequences up to approximately 170 kilobases using a modified bacterial artificial chromosome clone as the donor.

Yes! Conditional knockin mice are generated by inserting a mutation, reporter gene or tag that is not expressed until activated. One common conditional knockin strategy includes a loxP-stop-loxP cassette that silences the expression of the transgene or mutation until the stop cassette is removed by Cre recombinase. This approach allows tissue-specific and/or temporally controlled expression of the inserted sequence. Other conditional activation strategies such as the tetracycline regulatory system have also been successfully employed in mice.