Final Vectors
The EUCOMM final targeting vector is designed to generate a conditional mutation of a specific target gene via electroporation of mouse 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 to allow removal by 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 Cre recombinases.
The "critical" exon has been 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 alleles. That is, in the original version of the allele gene function is incativated by splicing of upstream endogenous exons to a splice acceptor in the targeting cassette (tm1a, see diagram below). 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).
The EUCOMM final vectors are distributed as circular plasmids. The plasmid have to be linearized before electroporation by an AsiSI restriction digest.
Promoter Driven Final Targeting Vectors
The typical EUCOMM targeting vector contains a targeting casssette 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, the βact-neo cassette and the critical exons are removed, resulting in a lacZ tagged null allele.
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.
