Point mutant gene knock-in models are models that have a point mutation in an endogenous gene which does not ablate the gene but merely changes its function. Inducible point mutation is a type of conditional knock-in strategy, which is commonly used to study the role of a lethal point mutation. At Creative Biolabs, both ESC/HR technique and CRISPR/Cas9 technique are provided for the generation of a conditional knock-in mouse model.
Technology Background
Conventional point mutant gene knock-in mice have been widely used and much can be learned from studying this kind of models, however, they have exhibited a number of limitations. First, it is not possible to conclusively ascribe a phenotype to a particular cell population or anatomic site. Second, the knock-in gene product could lead to developmental abnormalities and/or compensation in the expression of other genes, making it hard to determine if the observed phenotype is caused by the knock-in mutation or by developmental issues. Some mutant gene products could even be lethal. Thus, it would be highly advantageous to create animals that conditionally express the knock-in mutation in a tissue-specific and/or temporally controlled manner.
Inducible Point Mutation
Inducible point mutation models are defined as knock-in models where a specific point mutation is introduced into the sequence of the gene of interest at a particular time under application of inducers. This is achieved by using the Cre/LoxP system. In the absence of Cre recombinase, the engineered allele produces only wild-type product with no evidence of expression of the mutant. In contrast, following Cre-mediated recombination, only the point mutant product is produced.
Mechanisms of the Inducible System
To be specific, the induction is performed based on the existence of estrogen receptor (ER), a receptor that translocates from the cytoplasm to the cell nucleus to regulate gene expression after binding to the corresponding ligand, estrogen or tamoxifen (an estrogen antagonist). To eliminate the effect of endogenous estrogen, the binding domain of ER is artificially mutated and therefore it is only capable of binding to exogenous estrogen analogs, tamoxifen.
In this strategy, Cre is ligated to the ER and this fusion protein (e.g., CreERT2) will stay in the cytoplasm before the administration of tamoxifen, leading to functional expression of the wild-type gene. However, when tamoxifen is applied to the animal, either topically or through injection, it will bind to the ER as a ligand and thus activate the Cre-mediated recombination process. This way, the point mutation knock-in model can be obtained and timing control can be guaranteed.
Features
Developmental abnormalities and lethal phenotype of a conventional point mutation model can be avoided. Mutated protein is only expressed when inducers are given at a needed time of developmental stage. But a thorough analysis must be taken into consideration when designing the point mutation strategy to ensure that the knock-in mutation is expressed in an absolutely identical manner to the wild-type allele.
Other knock-in strategies that you may be interested include:
Creative Biolabs has constructed systematic data and experience in generating inducible point mutant mouse and rat models. Our research scientists will work closely with you to develop detailed study protocols to meet your goals. We are willing to apply our expertise in facilitating the brilliant studies of our clients.
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