Schematic of the ‘knockout-first allele
The initial allele (tm1a) contains a lacZ promoterless trapping cassette inserted into the intron of a gene, disrupting gene function. Flp recombinase converts the ‘knockout-first’ allele to a conditional allele (tm1c), restoring gene activity. Cre recombinase deletes the trapping cassette and floxed exon of the tm1a allele to generate a lacZ-tagged allele (tm1b) or deletes the floxed exon of the tm1c allele to generate a frameshift mutation (tm1d), triggering nonsense mediated decay (NMD)2 of the deleted transcript.
Promoter Driven Final Targeting Vectors
The "typical" 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.