A point mutation model from ingenious allows you to study a known disease-causing mutation or alter a critical site from the murine to human sequence. Different strategies are available 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.
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 consider a conditional point mutation strategy, described below.
Conditional point mutant lines don’t express the mutant sequence until you choose to activate it. Your tissue-specific Cre-expressing lines allow expression of the mutant gene only where Cre is active. Depending on your needs the allele can initially express the wild-type sequence or function as a null. Bypass the deleterious effects of your mutation on embryonic development and create a model where you control the timing and location of activation.
Advances in molecular biology and gene targeting now allow for generating animal 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, until Cre recombinase is activated. Cre deletes the wild-type sequence and allows expression of the mutant sequence.
The following are two design examples on how this can be accomplished:
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 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). Thus the mouse model is more versatile and can be utilized across scientific disciplines.
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 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.
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.
1) Rappaport A, Johnson L. 2014. Genetically engineered knock-in and conditional knock-in mouse models of cancer. Cold Spring Harb Protoc 2014(9): 897-911.
2) Rubinstein M, Japón MA, Low MJ. 1993. 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 21(11): 2613-7.