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Conventional Knockout Mouse Models

A faster way to knockout.

Conventional knockout mouse models have one or more genes inactivated in all tissues, at all times. Embryonic lethality is a possibility with conventional gene deletions, if the gene is crucial during development. The heterozygous mouse model may not present a phenotype, but in some cases the knockout can have an abnormal phenotype even in the heterozygous stage, such as with haploinsufficiency. Genes located on the X-chromosome need to be considered carefully because males will only have one copy of the gene and may be severely affected by the knockout.

Conventional knockouts offer a faster way to generate study-ready animals because no additional matings (e.g., mating with Cre mice) are needed. However, due to lethality risk and/or the gene being knocked out in all tissues rather than a specific tissue of interest, many researchers choose a conditional knockout approach instead. Below we discuss options for conventional knockout strategies.

Conventional Knockout Using CRISPR

Conventional deletions can be achieved using CRISPR technology. It is our mission to successfully generate your conventional KO mouse model as quickly as possible without sacrificing quality. Depending on the goal of your project, we can apply CRISPR by microinjection or via CRISPR-Assisted technology in ES cells. Please contact us to discuss which strategy would best meet your design parameters.

Conventional KO’s Using ES Cell Technologies

Gene Replacement with Neomycin Selection Cassette

Gene Replacement with Reporter

Knockout-First with Rescue of Expression

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Why is the Neo Selection Cassette Required?

Selection cassettes such as Neomycin, Hygromycin, and Puromycin are commonly used to generate mouse knockout and knockin models. The cassette serves important purposes during the mouse model production process, but it may cause issues in later stages of mouse development and affect the phenotype [1][2]. It is recommended to remove the cassette before studying the model. ingenious targeting laboratory has developed several proprietary FLP ES cell lines which aid to remove the selection cassette without any additional labor. This significantly speeds up the process of mouse model generation.

Here we discuss the utility of selection cassettes in mouse model development, define why they should be removed before studying the model, and provide examples of removal techniques.

In order to generate a mouse knockout or knockin model, recombinant embryonic stem (ES) cells with the correctly integrated targeting vector are injected into mouse embryos, which give rise to chimeric mice. To identify the correctly targeted ES cell clones, the selection cassette is required for two reasons:

Antibiotic Selection

Aid in Screening Strategies

How is the Neo Cassette Typically Removed?

The selection cassette is commonly flanked with FRT recombination sites, which are FLP recombinase recognition sites. In order to delete the selection cassette, the FLP recombinase enzyme must be introduced to recombine these FRT sites. This can be accomplished either in vitro via an additional electroporation step or in vivo via mating. In each case, additional labor is needed, resulting in longer project time lines and additional cost. Below we discuss both strategies in more detail, focusing on FRT/FLP recombination.

Additional Electroporation and Screening

Additional Mating


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