Targeting Vector Design

The basic targeting vector contains 5' and 3' arms of sequence homologous to the gene being targeted and positive/negative selection markers used to disrupt gene function and/or to identify ES cell clones that integrated targeting vector DNA following electroporation. Elements for the deletion of incorporated sequences at later stages of development or in specific tissues can be included. Inclusion of a point mutation that creates a different restriction pattern should be considered if necessary to distinguish integrated DNA sequence from endogenous sequences during ES cell clone screening.

General Consideration for Arms of Homology

There should be a sufficient length of homology, both 5' and 3' to the target gene, to permit efficient pairing and cross-over formation. While the length of the arms can vary, the homology of the two arms should be at least 5-8 Kb in total and the shorter arm no smaller than of 1 Kb in length. Targeting frequency may be enhanced when the genomic DNA of the arms is isogenic to the strain from which the ES cell line was derived.

Introducing Deletional Mutations (Knock-Out Models)

Mutations are introduced into the coding region to disrupt gene function by preventing transcription and translation of the targeted gene. This is often accomplished by replacing an upstream exon with a positive selectable marker, which also serves in the identification and enrichment of targeted clones.

Introducing Point Mutations (Knock-In Models)

Diseases are often caused by simple point mutations that alter gene function. Point mutations are initially created into a cloned exon by site-directed mutagenesis. Targeting vectors are designed with the selection marker placed in an intron flanked by loxP or FRT recognition sites. The endogenous exon is then replaced with the mutated exon/intronic marker and the selection marker is subsequently deleted from the genome via site-specific DNA recombination.

Positive Selection Markers

Positive selection is necessary to identify those ES clones that integrated the targeting vector following electroporation. The most common positive selection marker is the neo resistant (neor) gene. ES cells that incorporated targeting vector DNA with this marker continue to thrive when grown in the presence of G418 (Geneticin), while those that did not will die. Markers can be deleted from the genome at a later time if configured correctly in the targeting vector.

Negative Selection Markers

Negative selection can help distinguish clones that incorporate targeting vector DNA via homologous recombination (a site-directed targeted event) from those in which the DNA integrates randomly. Common negative selection cassettes include HSV-tk and PGK-DTÎ±, which are placed outside the regions of homology in the targeting vector. These markers are lost when the DNA integrates by homologous recombination but retained if integrated randomly. Theoretically, retention of these sequences in the randomly integrated DNA results in cell death. TK-mediated cell death requires the addition of the toxic DNA analog ganciclovir to the media. However, since this drug can cause non-specific cell death and does not typically result in a significant enrichment of targeted clones, we no longer subject clones to this selection. Presence of this marker in the targeting construct will not interfere with integration or selection, so removal of this marker from the targeting vector prior to electroporation is unnecessary. For the DTÎ± gene, expression of this toxin alone is all that is required to bring about cell death.