Figure 3.

Selection cassette recycling after homologous recombination. (a) An illustration of a typical homologous recombination event utilizing a positive/negative selection scheme. The gene sequence is shown with exons 2, 3, and 4. The targeting vector replaces exon 3 with a positive selectable marker, such as the PGK driven neomycin resistance cassette (phosphoglycerate kinase [PGK]-neo^R), and utilizes a negative selectable marker, such as the herpes simplex virus promoter driven thymidine kinase gene (HSV-TK), to counter-select against random integration of the targeting vector. Homologous recombination results in replacement of exon 3 with the PGK-neo^R cassette. (b) The use of site-specific recombinases such as Cre or Flp allows removal of a selection cassette before or after the production of an animal by SCNT. In order to accomplish this, positive marker is flanked by recombinase recognition sites like loxP. After homologous recombination (HR) the selection cassette can be removed by Cre recombinase in culture or in vivo. In order to select efficiently for removal of the selection cassette in vitro, a positive/negative selectable marker such as PuroΔTK or hygroCodA, with the negative selection marker outside the homology arms (for example, CodA, TK, or diphtheria toxin). (c) Schematic for generating conditional knockout alleles using site-specific recombinases Cre and Flp. A targeting construct is generated that leaves each exon intact, but includes loxP sites flanking an exon critical for gene function, in this case exon 3 (wild-type or an alternative allele). The selectable marker, flanked by frt sites, can be removed from the targeted allele either in vitro or in vivo to avoid selection cassette interference of the modified allele. Animals carrying this targeted modification can be crossed to animals that express Cre ubiquitously or in a specific tissue, resulting in progeny with a deletion of exon 3 in the whole animal or in a specific tissue.