(A) Generation of a targeted embryonic stem (ES) clones. ES cells have been shown as islands of cells growing on the top of mouse fibroblasts (feeder cells). The first step is the isolation of the genomic locus from the 129Sv library and construction of the targeting vector. Targeting vector has two flanking regions 1-2 kb and 5-7 kb, identical to the targeted locus; positive selection marker (neomycin, puromycin or hygromycin), placed between flanking regions; and negative selection marker (thymidine kinase gene), outside of the targeted sequence identity. After electroporation of ES cells with the targeted vector, homologous recombination will exchange the positive selection marker, i.e. neomycin, with the region, to be targeted. The positive ES clones will gain their resistance to the drug G418. The negative selection marker will decrease the number of non-homologous recombined colonies by the selection of ES cells in the presence of FIAU. ES cells after recombination could be screened either by PCR or Southern blot analysis, or both. The final proof of homologous recombination and number of integration sites should be always done by the Southern blot analysis. These steps are the most time and labour consuming and could take from 3 to 8 months, depending on the efficiency of homologous recombination in the desired locus.

(B) Injection of the targeted ES cells into donor blastocysts. This step usually takes 4-6 weeks. HP is designated for the holding pipette and IP - injection pipette accordingly. BC is the blastocoele, where targeted ES cells from the IP need to be injected. ICM is the internal cell mass, containing donor embryonic stem cells. Zona pellucida is marked ZP. Injected blastocysts are transferred to both horns of pseudopregnant foster mice, which are carrying them to term.

(C) Testing of chimaeric animal for the germ-line transmission (1-4 months). Chimaeric animals are scored by the agouti coat colour contribution. Four different chimaeras from 30 to 90% have been shown in the picture. Chimaeric animals with agouti contribution > 70% have the best chances for the germ-line transmission of the desired mutation.