Point Mutation

A point mutation mouse model is a substitution of one or more nucleotides with variant nucleotide(s). This can result in a frameshift following insertion or deletion mutations, or an in-frame amino acid change that thus changes the protein sequence of the gene of interest. There are various ways to introduce a point mutation into a mouse gene. At ingenious, we focus on specific gene targeting, so an engineered allele is expressed from a targeted locus according to our clients’ research interests.

A point mutation can be introduced into endogenous genes such that they are expressed conventionally, or in combination with elements that make them conditional. Here, we discuss our clients’ favorite conventional and conditional knockin approaches:

Conventional Point Mutation

A conventional point mutation results in the targeted allele only encoding the modified mutant protein.

Conventional Point Mutation from GeneTargeting.com

There is no additional mating required to study the mutation phenotype, except for generating homozygous animals if needed. Thus, results can be achieved quicker when compared to conditional mutations, where mating to Cre transgenic mice is necessary before the mutation is expressed. A conventional point mutation model is also simpler to generate compared to more complex targeted allele designs.

When conventional is not enough, our clients turn to more sophisticated mutation design options. Below, we discuss our top designs for accomplishing a conditional point mutation.

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.

Knockout-First with Conditional Activation of Mutant from GeneTargeting.com

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. In the schematic, an FRT (can be loxP instead) flanked STOP cassette is introduced in an intron upstream of the mutation. Depending on the specific mutation, the mutant protein may be truncated or fully inactivated. This can be assessed before the project is started.

 

Wildtype-First with Conditional Activation of Mutant

Advances in molecular biology and gene targeting now allow for generating mouse models with the ability to “turn on” a mutation in a specific tissue and/or at a specific time point. The gene will express as wild-type initially, and only upon Cre recombination will the mutant be activated.

The following are two design examples on how this can be accomplished:

Minigene Approach

Minigene Approach from GeneTargeting.com

Inversion Approach

Inversion Approach from GeneTargeting.com

There is a 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. Furthermore, a tissue or time specific mutation might 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 be used for this). Thus the mouse model is more versatile and can be utilized across scientific disciplines.

With advances in genome research and the development of new genetic engineering technologies, more exciting possibilities for gene targeting and mouse model generation will continue to emerge.

After You Receive Your Mice

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.

References/Further Reading

1) Rappaport A, Johnson L. Genetically engineered knock-in and conditional knock-in mouse models of cancer. Cold Spring Harb Protoc. 2014 Sep 2;2014(9):897-911.

2) Rubinstein M, Japón MA, Low MJ. Introduction of a point mutation into the mouse genome by homologous recombination in embryonic stem cells using a replacement type vector with a selectable marker. Nucleic Acids Res. 1993 Jun 11;21(11):2613-7.