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
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.
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.
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.
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.
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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
After the targeting vector is introduced into an ES cell line, the cells are treated with a specific antibiotic based on the selection cassette that was used for the targeting vector. Thus, clones that survive the antibiotic treatment have the selection cassette integrated into the mouse genome.
Aid in Screening Strategies
The second reason for using the selection cassette is to provide a screening strategy to identify ES cell clones that have the correct integration of the targeting vector. The selection cassette provides unique sequences and restriction sites that are used to design PCR primers and Southern blot strategies, to identify and confirm clones that have the targeting vector integrated into the correct location in the mouse genome.
Because the the selection cassette may interfere with gene expression and negatively affect mouse development [1], [2] it is recommended to remove the cassette before conducting studies with the mouse model.
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
The selection cassette can be removed right after the recombinant clones have been identified, with a second electroporation.
During the second electroporation, the site-specific recombinase FLP is introduced to delete the selection cassette. Chimera mice produced from those targeted, selection cassette-deleted ES cell clones are then mated with wild-type mice to produce the completed, germline confirmed mouse model.
There are drawbacks of using a second electroporation, however, such as the necessary extra manipulation and additional screening of the ES cells, longer project timeline, and additional labor required, and higher chances of reduced or absent germline transmission efficiency.
Additional Mating
A traditional way of removing the Neomycin selection cassette in vivo is by mating chimeric mice to FLP transgenic mice to introduce the FLP recombinase. In this workflow, chimera mice produced from targeted ES cell clones still carrying the selection cassette are mated with FLP recombinase mice to initiate the selection cassette deletion process. At minimum, a subsequent round of mating with wild-type mice is required to complete the deletion process and produce the completed, germline confirmed mouse model.
The drawbacks of adding FLP mating to the model production workflow include having to purchase and maintain FLP mice, the extensive time, labor, and costs devoted to the additional breeding steps and genotyping, and the need for detailed tracking and record keeping of the mice.
References
1) Hirotsune S, Fleck MW, Gambello MJ, Bix GJ, Chen A, Clark GD, Ledbetter DH, McBain CJ, Wynshaw-Boris A. 1998. Graded reduction of Pafah1b1 (Lis1) activity results in neuronal migration defects and early embryonic lethality.Nat Genet19(4): 333–9.
2) Xu X, Li C, Garrett-Beal L, Larson D, Wynshaw-Boris A, Deng CX. 2001. Direct removal in the mouse of a floxed neo gene from a three-loxP conditional knockout allele by two novel approaches. Genesis 30(1): 1-6.
