Recombination targeting construct

Many natural protein hgands bind to their receptors via interactions of a specific area of the protein backbone. The receptor binding domain of such a protein can be transferred into another protein, for instance a therapeutically active one. This technique is commonly applied in the preparation of recombinant targeting constructs, and will be discussed in Section 11.8.2. 

Development of Proteinaceous Drug Targeting Constructs Using Chemical and Recombinant DNA Approaches... 

Recombinant Domains as Building Blocks for Drug Targeting Constructs... 

Homologous recombination occurs approximately 1000-fold less frequently than non-homologous recombination, therefore methods have been developed to enrich for homologous recombination events. A widely applied method includes the use of a positive and a negative selection marker and does not require the expression of the target gene in ES cells (Mansour et al., 1988). The targeting construct is based on a replacement-type vector... 

When the targeting construct is planned, consideration should also be made as to how the homologous recombinants will be identified. As the most solid method to identify homologous recombinants, we recommend Southern blot... 

Fig. 4.1 Gene disruption by targeted mutation. To delete exon 2 (E2), a targeting construct was prepared with side arms containing homologous DNA upstream and downstream of exon 2, respectively. By recombination events in the upstream and downstream arms, exon 2 and part of the surrounding intronic region will be replaced by a neomycin selection cassette (neo), resulting in the inactivation of the gene.

Fig. 4.2 Detection of homologous recombinants by Southern blot. Restriction enzyme A cuts once in the homologous side arms of the targeting construct and once within the neomycin selection cassette. For Southern blot, the genomic DNA is digested with restriction enzyme A and hybridized with an external probe which stains bands of different size for homologous recombinants or the wild-type gene. In case of heterologous recombi...

In order to exclude the unlikely event that a heterologous recombination occurred together with the homologous recombination, a second Southern blot analysis is carried out with a probe inside the targeting construct (see Fig. 4.2). This internal probe will recognize not only homologous, but also heterologous recombinations. 

If the selection cassette also contains a negatively selectable marker such as tk, then cells that have lost the cassette can be selected for. The tk gene must be removed in vitro, as its expression impairs sperm motility in vivo and this results in sterile chimeric mice [21]. ES cells without a selection cassette can easily be retargeted by the original targeting construct to obtain homozygously recombined ES cells. 

The targeting construct will be electroporated into ES cells. After selection for stable transfectants expressing a specific antibiotic resistance (here neomycin resistance, neo), homologous recombinants will be identified and injected into blastocysts. Transfer of injected blastocysts into specially conditioned ( pseudopregnant") foster mothers results in chimeric mice that consist... 

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