A knockout mouse (KI mouse) has an element inserted into a specific locus in the genome. This can be accomplished with several strategies, including the use of the CRISPR/Cas9 system.
From single point mutations to the most intricate designs ingenious has delivered custom knockin mice again and again for 20 years. With a complete toolkit of different approaches and an experienced scientific staff, there is no knockin design that is out of reach. Contact us today to discuss the custom knockin mouse model your lab will use for years to come.
|Point Mutation||Cassette Insertions|
|| Single or multiple point mutations across regions big or small can be engineered for your project.
Targeted gene can only express mutant sequence.
|| Target your gene of interest with a cassette insertion to express an alternate sequence.
Cassette sequence can be expressed in addition to, or in place of gene’s sequence.
Can express Cre, reporter gene, human cDNA and more.
|| Bypass embryonic lethality with a conditional point mutation model.
Precisely control expression of your mutant gene sequence.
|| Enhance your model by taking advantage of the Cre/lox system.
Express cDNAs, reporter genes, tags, or a unique custom expression cassette in a conditional manner.
|Learn More||Learn More|
Humanized mice using ingenious’ TruHumanization™ strategy completely replaces endogenous mouse genes with the human genomic sequence including introns and regulatory features, and more. Leave nothing behind.
A reporter knockin mouse defines an animal model in which fluorescent, bioluminescent proteins or biochemical tags are inserted into the genome. The reporter can replace a gene, be fused to a protein or inserted into the 3′ UTR. Researchers utilize reporter knockin mouse models for drug screening, gene expression monitoring, knockout confirmation readout, biochemical analysis, and more. In generating these lines, researchers have to be aware that fusing a reporter to a protein may alter protein conformation, localization and functionality, but if fused via linkers, it will avoid disrupting the function of the target protein.
Tissue specific and doxycycline inducible/reversible expression control over your cDNA or multi component cassette has never been simpler. ingenious’ proprietary Inducible Rosa26-Express Targeting™ system provides a straightforward design option for those who require precise control for their research.
A proprietary cassette is inserted into the target gene, allowing you to create five different models including: global gene knockout, tissue/time specific rescue, and ectopic expression. Learn more about our F.A.S.T.™ technology!
To achieve cDNA knockin, ingenious offers two main strategies. One option is to replace the expression of your target gene with cDNA. This strategy in particular is versatile because it allows you to knock out a gene, while replacing it with your desired human sequence, reporter gene, and more. This is an ideal strategy for reporter lines as it ensures the most accuracy.
Another strategy is to co-express your gene of interest with the cDNA knockin. Because of this added flexibility, you can obtain reporter, Cre, and more lines with co-expression.
Click here for more information on cDNA Knockins at ingenious targeting laboratory.
To create a knockin mouse, a DNA construct must be made with the insert sequence flanked on both sides by sequences from the mouse genome. The flanking sequences target the insertion to the desired location by inducing homologous recombination. The knockin mice method is different from classic pronuclear injection transgenesis, where genes are inserted randomly into the mouse genome.
DNA can be introduced directly into mouse embryos or embryonic stem cells and will integrate at a low frequency, creating transgenic embryos or cells. Variations on this technique, including the use of the CRISPR/Cas9 system, increase the frequency of integration and can enable creation of targeted transgenic mice lines.
When thinking about a design for a new knockin mouse model, you should first check and see if your desired model already exists. Once you’ve looked through several online databases and determined that the model you need is not already available, you will have to decide if your experiment calls for random or targeted insertion. Here at ingenious, we specialize in targeted insertion, which will give your knockin mouse the predictability you may be seeking. While random insertion is usually available at a lower cost, it requires extra validation on your end to determine the exact location where the transgene was inserted. This may also make it difficult to evaluate if it’s an accurate knockin mouse model of the disease and gene you are studying. That’s why targeted transgenic mice are often the model of choice when studying specific mutations and diseases.
One other consideration to keep in mind is whether the knockin may or may not be lethal to the mouse. Fortunately, there’s a way to get around this. By making your knockin conditional, you have the ability to turn the mutation on when you need to observe it. This can be achieved with tissue specific or temporal control. In addition to targeted transgenics, ingenious is capable of creating conditional knockin mice as well.
To prepare for your experiment, you may also want to consider creating a breeding plan before you receive your mice. ingenious makes this easy with our very own, free Breeding Planner. Preparing for colony management ahead of time can help you make the most out of your new knockin mouse line.
For more information, see our guide “3 Things I Wish I Knew Before Making My First Knockin Mouse Model.” If you are still unsure of what type of strategy may be the best fit for your new knockin mouse model, please contact us and one of our scientific consultants will be in touch with you to discuss your research project.
For a client investigating Cofilin-2 (CFL2) mutations in humans and their association with myopathies, such as nemaline and myofibrillar myopathy, ingenious generated a Cfl2A35T/A35T knockin mouse. These mice appeared the same as WT mice, however the myopathy progressed much faster, resulting in the death of Cfl2A35T/A35T mice by the end of the study’s 9th day. Furthermore, the knockin mice were found to have lower levels of Cf12 mRNA in tissues throughout the body, along with alternate splicing and two alternate transcripts. This suggests that pre-translational splicing could be associated with CFL2 mutations and their relationship with the development of myopathies.
By using a homologous recombination-based technique, scientists at ingenious identified a positive C57BL/6N BAC clone (RPCI-23) clone, allowing the construction of a targeting vector by the use of a ~8.9 kb region. A point mutation was then created in exon 2 through PCR, after adding a pGK-gb2 loxP/FRT-flanked Neo cassette downstream of exon 2. The construct’s total size was ~11.3 kb. To remove the Neo cassette and create heterozygous mice, resulting chimeras from the microinjection of IC1 C57BL/6 ES cells were mated with WT C57BL/6 FLP homozygous mice.
Rosen SM, Joshi M, Hitt T, Beggs AH, Agrawal PB. 2020. Knockin mouse model of the human CFL2 p.A35T mutation results in a unique splicing defect and severe myopathy phenotype. Hum Mol Genet.
ingenious generated an Arc knockin mouse for a client, to study the relationship between arc expression and cognitive flexibility. To create this mouse, two point mutations were performed in iTL IC1 (C57BL/6) ES cells, located at the Arc gene’s Exon 1. The goal of this was to substitute Lysine in place of Arginine in positions 268 and 269. After screening and microinjecting positive clones, chimeras were mated to C57BL/6 FLP mice as well as backcrossed five times to C57/BL6 mice, resulting in ArcKR/KR (ArcKR) mice.
Through the use of this model, our client was able to observe that ArcKR mice experienced some loss in reversal learning, compared to Arc+/+ (WT) mice. However, their spatial learning abilities were about the same. This highlights the importance of Arc signaling pathways, as well as other activity-dependent molecules, in both reversal learning and overall cognition.
Wall MJ, Collins DR, Chery SL, Allen ZD, Pastuzyn ED, George AJ, Nikolova VD, Moy SS, Philpot BD, Shepherd JD, Müller J, Ehlers MD, Mabb AM, Corrêa SAL. 2018. The Temporal Dynamics of Arc Expression Regulate Cognitive Flexibility. Neuron 98(6): 1124-1132.e7.
By generating a conditional point mutation mouse model of lymphoma with ingenious, one of our clients at Columbia University was able to make an important contribution to cancer research. A conditional point mutation was necessary for this type of model in order to accurately represent the RHOA G17V mutation that causes angioimmunoblastic T cell lymphoma (AITL), rather than gene loss that may be seen in a cKO. With this, our client discovered therapies that could help stop the growth of cancerous tumors.
Cortes JR, Ambesi-Impiombato A, Couronné L, Quinn SA, Kim CS, da Silva Almeida AC, West Z, Belver L, Martin MS, Scourzic L, Bhagat G, Bernard OA, Ferrando AA, Palomero T. 2018. RHOA G17V Induces T Follicular Helper Cell Specification and Promotes Lymphomagenesis. Cancer Cell 33(2): 259-273.