Figure 1. Knock-in targeting constructs. To illustrate the knock-in strategy, the genomic organization of a mammalian gene with four exons is shown in (a); (b)–(e) represent different constructs in order to perform a knock-in of a marker such as LacZ (b) or of any other cDNA of interest (gene X in (e)) into the locus (a); (b)–(d) illustrate the steps that are required to achieve a knock-in experiment (shown for LacZ). The first exon containing the ATG is deleted and replaced by a DNA fragment consisting of LacZ and neo (b). LacZ contains promoterless coding sequences including ATG and a polyadenylation site. The neo is inserted just behind the LacZ and the PGK promoter drives its expression. In a first electroporation experiment, the targeting construct (b) is introduced into wild-type ES cells. Homologous recombination events generate ES clones that carry a knock-in allele, as shown in (c). A transient expression of the Cre recombinase (also driven by the PGK promoter) in these ES clones to remove neo is achieved through a second electroporation. The genomic structure of the final knock-in allele is shown in (d) for LacZ or any other cDNA in (e). LacZ or gene X is then expressed under regulatory sequences of the locus (a). (f) represents a targeting construct for the generation of a floxed allele by using the Frt sites (F) to delete the selection marker. The neo gene is flanked by two Frt sites (in parallel orientation) and carries in addition one LoxP site (arrowhead). The second LoxP site required for the floxed allele is inserted in front of the first exon. The Frt sites allow the excision of the neo gene by crossing mice carrying the mutated allele in (f) to transgenic mice that express the FLP recombinase (Dymecki, 1996). The FLP recombinase is expressed under a ubiquitous promoter such as Rosa26. After deletion of the neo, the LoxP and one Frt site remain in the locus (g). (g) represents the genomic organization of a floxed allele generated from the gene (a), where LoxP sites are flanking the first exon. Transgenic mice expressing Cre recombinase driven by specific promoters may be used to knock out gene (a) (using the floxed allele) in specific tissues. External 5’ and 3’ probes for the screening of homologous recombination events are indicated in (a). The insertion of LacZ or cDNA (X) can also be designed in such a way that a fusion protein between the endogenous and the inserted gene is created. Any exon may then be used for the creation of the fusion protein. neo, neomycin; -gal, -galactosidase; PGK, phosphoglycerate kinase; Gene-X may represent any cDNA to be knocked in the locus. The arrowheads show the LoxP sites that can be recognized by the Cre recombinase to excise the neo gene.

Figure 2. Mouse preimplantation development. (a) Fertilized oocyte with the male and female pronuclei. (b) and (c) illustrate the two- and four-cell stages, respectively. (d) and (e) show the morula and compacted morula, respectively. (f) A blastocyst, shown in (g) at high magnification to indicate the different cell types: TE is the trophectoderm, which will give rise to extraembryonic tissues, and ICM is the inner cell mass from which the embryo proper is derived. The ICM are the cells giving rise in vitro to ES cells. ZP, zona pellucida. Polar bodies are not shown.

Figure 3. Insertion of the -galactosidase gene into the Pax-3 locus. Staining for -galactosidase activity in whole-mount embryos from E10.5 of gestation. The -galactosidase gene, inserted into the Pax-3 locus by homologous recombination in ES cells, recapitulates the expression of the Pax-3 gene in vivo. Pax-3 is a paired box-containing gene and a member of the Pax family of transcription factors. The expression is clearly seen in the spinal cord (SC) and in the developing skeletal muscle. (a) A heterozygous embryo. The muscle cells are regular and migration of these cells into the limb buds is occurring normally (arrows). (b) A homozygous embryo reveals LacZ staining in irregular forming muscle precursor cells (black arrows); note that muscle cells are not migrating into the limb buds (white arrow). In the posterior part of the spinal cord, the neural tube is open, leading to spina bifida (Sb). Figure 3b is taken from Mansouri et al. (2001) Development 128: 1995–2005.