Designing a targeting vector for proper detection

SOM Home > Research > Gene Targeting and Transgenic Facility > Embryonic Stem Cell > Designing a targeting vector for proper detection

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

koscreen2

ut = untargeted

T= targeted

Both probes hybridize with a 9.95 kb band at the wild type allele.

 

 

For the 5' probe, the targeting event replaces the pink exon with the blue exon containing the restriction site, creating a 5.1 kb band.

For the 3' probe, the targeting event replaces the pink exon with the blue exon containing the restriction site, creating a 6.14 kb band.


The second example deletes a restriction site

koscreen

ut = untargeted

T= targeted

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.