Custom Gene Targeting Vector Synthesis

Gene targeting can be used to generate knockout, knockin or conditional alleles. 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 positive cell clones that have integrated targeting vector DNA following transfection.

A targeting vector needs to contain a sufficient length of homology, both 5′ and 3′ to the target gene, in order to permit efficient homologous recombination. 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 DNA sequence of the arms is isogenic to the strain from which the ES or somatic cell line was derived.

Positive selection is necessary to identify the targeted cells that have integrated the targeting vector following transfection. The most common positive selection marker is the neomycin resistant (Neo) gene. Negative selection can help distinguish clones that incorporate targeting vector DNA via homologous recombination from those in which the DNA integrates by random insertion. Common negative selection cassettes include HSV-tk or PGK-DTA, which is placed outside the regions of homology in the targeting vector. These markers are lost when the DNA integrates by homologous recombination but retained if inserted randomly. Retention of these markers results in cell death.

BiCell Scientific’s gene targeting vector synthesis service includes genomic DNA synthesis, molecular cloning, targeting sequence assembly, and Sanger sequencing for validation.