Vectors

Final Vectors

The EUCOMM final targeting vector is designed to generate a conditional mutation of a specific target gene upon electroporation (homologous recombination) in mouse embryonic stem (ES) cells.

The main components of a EUCOMM vector are 5′ and 3′ homology arms that mediate homologous recombination, and a central targeting cassette that disrupts gene function and reports gene expression with a lacZ reporter. The targeting cassette is flanked by FRT recombination sites, which allows to remove the targeting cassette with Flp recombinase. In addition, the vector introduces a pair of loxP recombination sites around a "critical exon", which is the prerequisite for conditional gene inactivation by deleting the "critical exon" with Cre recombinase.

The "critical" exon is chosen by the EUCOMM vector design team as an exon that is present in all splice variants of a gene, and which upon removal causes a frame shift, leading to complete gene inactivation.

The alleles generated after targeting with a final vector are so-called "knock-out" first or "targeted trap" alleles. In the original version of the allele (tm1a) gene function is inactivated by splicing of upstream endogenous exons to a splice acceptor in the targeting cassette. To render this allele into a conditional version, the targeting cassette has to be removed by Flp recombinase, which leaves loxP sites flanking a "critical exon" behind (tm1c). This conditional allele can then be mutated subsequently by Cre recombinase in a time and tissue-specific manner (tm1d). Alternatively, the original allele can be treated with Cre recombinase to remove the critical exon, which generates a lacZ-tagged null allele (tm1b).

For more information, see also EUCOMM/EUCOMMTools Nomenclature

Promoter Driven Final Targeting Vectors

The typical EUCOMM targeting vector contains a targeting cassette with a neo-resistance gene that is driven by the β-actin promoter. This allows targeting of all genes, irrespective of their expression status in mouse ES cells. The example below shows a promoter driven vector for the targeting of the Sema3a gene. The allele can be rendered conditional by subsequent excision of the targeting cassette by Flp recombinase, either by transfection of ES cells with a Flp plasmid, or by breeding with a Flp-deleter mouse strain. When applying Cre recombinase to the original version of the allele (tm1a), the βact-neo cassette and critical exons are removed, resulting in a lacZ tagged null allele.

Promoter Driven Final Targeting Vectors

Promoterless Final Targeting Vectors

The promoterless final targeting vectors can be used for the targeting of genes that are expressed in ES cells at sufficient levels. This strategy utilizes the promoter of the targeted gene to drive expression of the neo resistance of the targeting cassette (see example of Gsk3a promoterless vector below). Similar to alleles generated with promoter driven vectors, the targeting cassette can be removed by transfection of ES cells with a Flp plasmid, or by breeding with a Flp-deleter mouse strain. A lacZ tagged null-allele can be generated by deleting the critical exon using Cre recombinase.

Promoterless Final Targeting Vectors

Intermediate Vectors

Intermediate vectors are precursors of final vectors and lack the central targeting cassette. They are distributed to users who want to assemble their own targeting vectors with specific features that are not present in the EUCOMM final vectors.

The figure below illustrates the final steps leading from the intermediate vector to the final targeting vector. The intermediate vector contains the 5’ and 3’ homology arms for homologous recombination and the critical exon. One loxP site (red triangle) is also present. The intermediate vector still lacks the targeting cassette and a backbone with the negative selection cassette (DTA).

Intermediate Vectors

EuMMCR distributes intermediate vectors to interested users, who would like to introduce the targeting cassette of their liking into the final targeting vector, e.g. fluorescent reporters (GFP, mCherry, etc.), dominant alleles, suicide genes (DTA), or any other gene cassette for ectopic expression.

It is not possible to use intermediate vectors directly for the generation of mutant ES cell clones via electroporation, since they are lacking key features of the final targeting vectors.