VelociGene is a novel method developed by Regeneron scientists which has revolutionized the process of gene targeting in ES cells. This method allows for a dramatic increase in the speed and throughput at which targeted ES cells can be generated, while at the same time increasing the precision, flexibility and complexity of the genetic modification. VelociGene achieved these improvements by incorporating two enabling innovations:
1. BacVec Targeting Vectors
VelociGene's use of BacVecs has greatly streamlined the process of making a knockout mouse while providing maximum flexibility for allele design. After obtaining a BAC clone of the target gene, we use bacterial homologous recombination to construct the knockout or other allele. The modified BAC is simply linearized, without additional trimming, to create the BacVec targeting vector prior to introduction into ES cells. In addition to simplifying construction, the long homology arms of BacVecs promote efficient homologous recombination in ES cells and enable larger deletions in a single targeting step than are possible with conventional targeting vectors.
2. The Loss of Allele Screening Assay
To take full advantage of BacVecs as targeting vectors we developed a proprietary Loss of Allele (LOA) assay, which employs quantitative PCR (Q-PCR) to detect the loss of one of the native target gene alleles upon recombination with the BacVec. We complement the LOA assay with a Q-PCR that quantifies the number of copies of the inserted reporter-selection cassette, valuable information not usually obtained by standard screening methods. For a correctly targeted ES cell clone the LOA assay indicates loss of one allele and gain of one copy of the reporter-selection cassette.
In developing VelociGene our goal was to bring to the process of knockout mouse production the speed, reliability, high throughput, and scalability of an industrial assembly line while retaining the precision and flexibility in allele design of a custom research project.
For inquires regarding VelociGene licensing or partnership opportunities, please contact us at business.development@regeneron.com.
