Figure 1. Scheme of ES cell technology. (a) The establishment of ES cells from the mouse blastocyst is schematically shown. The brown colour indicates that the ES cells are isolated from a mouse strain with an agouti coat colour which is dominant over albino (white) or black. This is important to monitor the degree of chimaerism by the coat colour (see also (b)). (b) Generation of chimaeric mice by blastocyst injection or morulae aggregation. The aggregated embryos are transferred to foster mothers. When the born embryos are partially derived from the pluripotent ES cells, the coat colour is mixed, as schematically shown by the brown stripes. These mice are called chimaeras. When they are mated to normal wild type mice they often do transmit the ES cell genome to the germline. (c) A typical gene targeting experiment is indicated. The construct is electroporated into ES cells. After selection and screening, the positive homologous recombinant clone (shown in blue) is aggregated to wild type embryos to generate chimaeras, which transmit the mutation to the mouse germline. The mutation is schematically indicated in the blow up on one chromosome with *. ICM, inner cell mass and TE, trophectoderm.

Figure 2. Targeting vectors. There are two types of targeting vectors which are depicted in (a) and (b). (a) Represents replacement-type targeting vector which is linearized outside of the region of homology. The vector sequences are collinear with sequences of the endogenous gene. Homologous recombination between genomic and targeting vector sequences leads to the replacement of a portion of locus of interest with targeting vector sequences. Sequences lying outside of the homologous region are excluded from the integration. Exons are shown in boxes and labelled by letters. Broken lines indicate plasmid sequences. (b) Represents an insertion-type vector which is linearized within the region of homology to endogenous sequences. The homologous recombination at the double-strand break in exon 2 results in the insertion of the whole vector, including plasmid sequences (broken lines), and leading to a partial duplication of the targeted gene. Numbers indicate exons; sequences homologous to the gene to be targeted are represented by a thick line, letters indicate restriction enzyme sites. Neo is the selection marker neomycin resistance gene.

Figure 3. Conditional gene inactivation procedure. A gene targeting experiment using the Cre/LoxP and Flp/Frt recombination systems is schematically shown. Exons are indicated by closed boxes and labelled from E1 to E5. (a), the wild-type allele; (b), the targeting construct, where the neomycin resistance gene is inserted in the second intron. It is flanked by the Frt sites and contains one LoxP site (black arrow); A second LoxP site is inserted into the first intron. (c), the mutated allele after homologous recombination. At this stage chimaeric mice are generated and the neomycin gene is removed after introducing the mutation into the mouse germline. This is done by crossing the floxed mice to transgenic mice expressing the Flp recombinase under the control of a ubiquitous promoter (such as Rosa26Flp mice). Homologous regions to the that participate in recombination are indicated. In targeting construct, HSVTk is the thymidine kinase gene used for the negative selection with ganciclovir (cells which have integrated the tk gene do not survive selection with ganciclovir). neo, neomycin (cells which have integrated the neomycin resistance gene survive selection with geneticin (G418)).

Figure 4. Site-specific recombination in mice. In first step a knockout mouse is generated where the gene of interest is functional but flanked by LoxP sites ‘floxed allele’ (mouse b). Before performing the specific gene inactivation, the Cre mice that will be used are crossed to the knockout mice (generated from the floxed mice by crossing to ubiquitous Cre), to get transgenic mice expressing Cre and one knockout allele (+/–), as shown for mouse a. When a female mouse b is crossed to a transgenic male mouse a, expressing a Cre transgene driven by a specific promoter (P), and carrying one knockout allele of the gene of interest, mice that are born exhibit the genotypes depicted in the animals c, d, e and f. The desired specific inactivation occurred in mouse c. When the Cre recombinase is used with an inducible system (hormone or tetracycline) the inactivation of the gene can be performed in a specific organ or tissue at a certain stage of development or at a postnatal stage. The same strategy could be followed using an inducible Cre that is driven by a ubiquitous promoter. fl, floxed; +/–, heterozygous for the null knockout allele.

Figure 5. Gene trap and promoter trap vectors. The principle of Gene Trap and promoter trap strategy is outlined in (a, b and c) and (d, e and f), respectively. In both cases a fusion transcript is the result of the insertion mutagenesis and leads to a fusion protein of the coding sequences of the trapped gene and the reporter (c and f). This allows the expression pattern of the new identified gene to be monitored. In addition, its function is inactivated and one can proceed for its cloning by PCR-based techniques. The selection marker, the neomycin resistance gene may be used with an independent promoter or can be integrated as a fusion protein to the -galactosidase gene (-gal) (not shown here). In this case only those genes are accessible to trapping that are expressed in undifferentiated ES cells. Usually strong promoters such as PGK or -actin are used to control neomycin expression. Px represents the coding sequences of the trapped gene.