Conventional Knockout Mouse Models

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.

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.

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.

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.

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.

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.

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.

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.