Point Mutation Mouse Models

Precise targeted knockin for your research

The point mutation mouse models offered by ingenious targeting laboratory provide researchers with precise tools for studying known disease-causing mutations or altering critical sites from murine to human sequences.

Examples of design approaches include conventional point mutation knockin, where the targeted allele encodes only the mutant sequence, or conditional designs, such as the minigene and inversion methods, allowing for activation of a mutation in specific tissues or at specific time points.

What Researchers Say

"iTL has generated three knockout and conditional KO mice for my lab. One of them is now published and we would like to thank iTL for the very nice service."

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— Kai Ge, PhD, NIDDK, National Institutes of Health

Point Mutation Mouse Models Overview

Point mutation mouse models from ingenious allow you to study a known disease-causing mutation or alter a critical site from the murine to human sequence.

Different strategies are available for creating point mutation mouse models depending on the experiments you have planned. No design is out of reach – our experienced staff will work with you to identify the best options for your gene of interest.

Conventional and Conditional Point Mutation Knockin Approaches

Conventional Point Mutation Knockin

A conventional point mutation results in the targeted allele encoding only the mutant sequence. This can be a powerful model for human disease, for example by changing a crucial codon to cause a deleterious amino acid substitution.

If you want more control over expression of the mutant sequence in your point mutation mice, consider a conditional point mutation strategy, described below.

Conditional Point Mutation Knockin

Conditional point mutation mouse lines don't express the mutant sequence until you choose to activate it. Your tissue-specific Cre-expressing mouse lines allow expression of the mutant gene only when or where Cre is active.

Depending on your needs the allele can initially express the wild-type sequence or function as a null. Use this to bypass the deleterious effects of your point mutation on embryonic development and create a model where you control the timing and location of activation.

Wildtype-First with Conditional Activation of Mutant

Advances in molecular biology and gene targeting now allow for generating animal models with the ability to "turn on" a point mutation in a specific tissue and/or at a specific time point.

The gene will express as wild-type initially, until Cre recombinase is activated. Cre deletes the wild-type sequence and allows expression of the mutant sequence.

Benefits:

• Lower risk of embryonic lethality or artificial phenotype since the mutation would be activated in only a specific tissue, or at a specific time point
• A tissue or time specific mutation may more accurately reflect the disease of study
• With the variety of Cre transgenic mouse lines available, multiple lines can be generated from the parental targeted mouse line, for expressing the mutant in different tissues or at specific times (CreERT2 or tetO-Cre can also be used for this)
• The point mutation knockin mouse model is more versatile and can be utilized across scientific disciplines

Point Mutation Knockin by Minigene Approach

The minigene approach involves introducing a construct that contains both wild-type and mutant sequences, separated by LoxP sites. Before Cre activation, the wild-type minigene is expressed. After Cre-mediated recombination, the wild-type sequence is excised and the mutant sequence is expressed.

Notable Client Publications

Cortes JR, Ambesi-Impiombato A, Couronne L, Quinn SA, Kim CS, et al. 2018. RHOA G17V Induces T Follicular Helper Cell Specification and Promotes Lymphomagenesis. Cancer Cell 33 (2): 259-273.

Kim E, Ilagan JO, Liang Y, Daubner GM, Lee SC, et al. 2015. SRSF2 Mutations Contribute to Myelodysplasia by Mutant-Specific Effects on Exon Recognition. Cancer Cell 27 (5): 617-630.

Point Mutation Knockin by Inversion Approach

The inversion approach uses an inverted mutant sequence that is flanked by lox sites oriented in opposite directions. Before Cre activation, only the wild-type sequence is expressed because the mutant sequence is in the wrong orientation. After Cre-mediated recombination, the mutant sequence is inverted into the correct orientation and expressed.

Notable Client Publications

Venturutti L, Teater M, Zhai A, Chadburn A, Babiker L, et al. 2020. TBL1XR1 Mutations Drive Extranodal Lymphoma by Inducing a Pro-tumorigenic Memory Fate. Cell 182 (2): 297-316.e27.

Knockout-First with Conditional Activation of Mutant

In some cases, our clients require more sophisticated mutation design options to allow for inducible expression of their mutation. One method that has been very successful and widely adapted is the use of removable transcription termination elements to control the expression of targeted alleles.

Excision of the STOP cassette, and resulting expression of the mutant gene, is mediated by inducible and/or lineage specific recombinases (e.g., Cre or FLP), which allows spatial and temporal control of gene expression.

Generating knockout-first with conditional activation

Generating a knockout-first mouse model with the ability to activate the mutant gene expression in a tissue specific or temporal manner via FLP or Cre recombination allows for producing two mouse models from a single gene targeting event.

The knockout-first approach is particularly useful if a global knockout has not yet been generated. With this strategy a STOP cassette is introduced in an intron upstream of the mutation. A recombinase deletes the cassette, allowing expression of the mutant gene.

What do I need to consider when creating a knockin mouse?

Combining your point mutation model with additional mouse lines to generate age matched cohorts can be challenging. ingenious' post project support services aim to simplify the process.

Utilizing our proprietary prediction and modeling software, our team of experts can generate the cohorts you need within projected timelines and costs that best fit your lab.

Frequently Asked Questions

What is a point mutation example?

A good example of what a single point mutation can lead to happens in the case of converting the GAG codon into GUG. This would lead to the encoding of amino acid valine instead of glutamic acid.

What is the difference between knockout and knock-in mice?

A point mutation occurs in a gene when a single base pair is added, deleted or changed. While most point mutations are harmless, some can have various functional consequences, including changes in gene expression or alterations in encoded proteins which can lead to disease.

Related Pages

• Knockin Mouse Models
• cDNA Knockin