Embryonic Stem Cell Lines Technology from GeneTargeting.com

 

Conventional Knockout Mouse Models

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 mouse models 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 Mouse Models from GeneTargeting.com

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Gene Replacement with Neomycin Selection Cassette

The target region of interest is replaced with the Neomycin selection cassette, which is also used for positive selection of targeted ES cell clones during tissue culture. As a result, the gene should be permanently inactivated at all times, in all tissues. The Neo cassette can be retained in the mouse model to act as a potential gene-blocking “trap” within the gene locus that it was inserted into. Alternatively the cassette can be deleted via FLP recombination in vitro (using FLP ES cells) or in vivo (via mating to FLP transgenic mice). Because the Neo cassette can potentially disrupt other genes nearby, many researchers prefer to delete it.

 

Strategy Considerations & Schematic

  • Delete all or most of the gene’s coding region – we are able to delete up to 100kb using large BACs

  • Delete promoter related sequences together with at least the first coding exon, preferably more

  • Delete an important domain without which the protein cannot function

  • Delete any part of the gene as specified by the client

  • Aim to create a coding frameshift if only a portion of the gene is deleted

Gene Replacement with Neomycin Selection Cassette from GeneTargeting.com

Gene Replacement with Reporter

The target region can be replaced with a reporter gene, such that the reporter gene expression will be controlled by the target gene’s endogenous promoter. This will enable the researcher to visualize where the gene is normally expressed. The same risks apply as for the traditional conventional knockout strategy described above. Furthermore, the reporter may not be detectable if the endogenous promoter is weak. Some fluorescent reporters have been shown to fluoresce more strongly or be more readily detectable than others, for example BFP and tdTomato.

Carefully studying technical data sheets of these reporters, and consulting with the companies that sell them and others who are familiar with using reporters will be useful. The selection of the appropriate reporter gene to use should also depend on the detection instrumentation to be used, the nature of the biological sample to be studied (e.g., tissue versus cells), and the type of experimental assay.

 

Strategy Considerations & Schematic

The reporter, including a polyA signal, is typically inserted at the endogenous ATG start site of the target gene of interest, usually also replacing the first exon of the gene. All other exons and introns can be kept intact to avoid deleting regulatory or promoter associated regions important for transcription from the target gene, for expressing the inserted reporter gene.

Some researchers prefer to not delete any endogenous gene sequence to avoid deleting important promoter related elements. In this case the reporter can be inserted at the ATG without replacing any gene sequence. The polyA signal included with the reporter gene should prevent read-through to avoid expression of the endogenous gene.

Gene Replacement with Reporter from GeneTargeting.com

Knockout-First with Rescue of Expression

By inserting a loxP flanked strong stop cassette in an early intron of the gene, the gene can be inactivated in a conventional manner. Utilizing Cre recombination to remove the stop cassette, the gene’s expression can be rescued in a tissue specific, temporal or global manner, based on the specific Cre used. In addition, a reporter gene can be included to express when the gene is inactivated. The reporter can be deleted along with the stop cassette, or alternative, custom design options can be incorporated.

Knockout First with Rescue of Expression from GeneTargeting.com

 

Our proprietary F.A.S.T.™ system can be used to produce a global knockout-first model, due to the stop component within the F.A.S.T. cassette. Expression can be rescued via Cre recombination, as well as inducible and reversible gene expression possibilities using the Tet system.

Conventional Knockout Using CRISPR

Conventional Knockout Using CRISPR from GeneTargeting.comConventional 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.

After You Receive Your Mice

Crossing your conventional knockout mouse model with additional mouse lines to generate cohorts can be challenging, especially in the absence of proper planning. ingenious’ post project support services aim to make the process easy and straightforward. Utilizing our proprietary prediction and modeling software, our team of experienced scientists craft plans for generating the cohorts you need based on timelines and costs that best fit your lab.