Figure 1

Gene targeting strategies used in mouse ES cells. Targeting is achieved by recombination (black crosses) between homology arms (red lines). (a) A knockout vector replaces an entire gene with a selection cassette containing drug resistance (D^R), enabling the selection of successfully targeted ES cell clones. (b) A knock-in vector allows the expression of a transgene, such as LacZ or Cre, by the promoter (gray arrow) of the targeted gene. (c) Insertion vectors can interfere with splicing by disrupting a target gene by the introduction of an exon with an early termination codon or a 5' splice acceptor site (SA). They typically target the genome with a single crossover event. (d) A conditional allele with directional DNA sequences (LoxP, green triangles) either side of a critical exon. Recombination between the sites will result in a null allele. (e) LoxP sites can also be targeted megabases apart, either side of a larger cluster of genes, enabling chromosome engineering. (f) Heterospecific Lox sites, such as LoxP and Lox511, are targeted by the site-specific recombinase Cre. Recombinase-mediated cassette exchange (RMCE) enables the efficient swapping of one targeted cassette containing incompatible target sites for another cassette flanked by an identical pair of sites. This enables the rapid generation of new alleles, such as introducing a point mutation in a critical exon.