Designing a Targeting Vector for Proper Detection
Design of the targeting construct is important to allow screening for targeting construct insertion at the homologous site
In both cases, it is important to have probes that are specific for sequences outside of the targeting vector. This is so that it will detect sequences at the correct endogenous site rather than wherever the targeting construct may integrate.
The first example inserts a restriction site
ut = untargetedT= targeted
Both probes hybridize with a 9.95 kb band at the wild type allele.
The second example deletes a restriction site
ut = untargeted
For the 5′ probe, the wild type allele makes a 4.1 kb band. For the 3′ probe, the wild type allele makes a 5.0 kb band
For both probes, the deletion of the restriction site (in the pink box) and replacement with the blue sequence lacking the site makes an 11.1 kb band.