General procedure for generation of a conditional knockout mouse strain by gene targeting strategies. (A) Generation of a targeting vector containing critical exon of targeted genes (red rectangle), two loxP site (green triangle), a positive (+) selection cassette and sequences of homology with the target locus (blue line). (B) The vector is linearized and electroporated into ES cells. (C) Correct tranformants are selected for in the presence of a selectant (eg. G418 if a neomycin resistance cassette is present in the targeting vector). (D) Correctly targeted ES cell clones are then identified and genetically characterized using long range PCR or Southern blot analysis. (E) The selected ES cell clones are then microinjected into 3.5 dpc blastocysts and transplanted into the uteri of pseudopregnant females. (F) Chimeras obtained from the microinjections are mated with wild-type mice to establish germ-line transmission of the modified allele. (G) Progeny derived from the chimeras are characterized using long range PCR or Southern blot analysis, and a mutant mouse line that carries the desired targeted allele is established.