Screening
Effective and accurate vector integration is the most important step to obtaining germline transmission. Confirming that this integration has been successful provides you with the confidence that your custom mouse model has achieved an important milestone towards completion. iTL offers a comprehensive, multi-step approach in identifying and confirming that this has been achieved and resulted in positive cell clones.
The screening process is divided into two phases. The first phase, PCR screening, establishes guidelines for the more thorough second phase, Southern blot screening. The combination of both PCR and Southern assays provides the confidence and security of accurate identification of your targeted positive clones.
PCR Assay Screening
DNA extractions from clones are used as target templates. Target specific primers allow detection of either the transgene or a knockout gene. The PCR reaction results in amplification of expected size fragments allowing for the determination of the genetic nature of the clones.
Southern Blot Screening
Clone DNA is digested with restriction enzymes to produce fragments which are separated using an agarose gel and blotted onto a specialized membrane. This membrane is hybridized with custom probes designed for your gene target(s). Signal and image development provides a banding pattern to analyze for confirmation of correct vector integration.
After positive clones are identified and expanded, they are confirmed again for successful recombination prior to delivery. You are provided a comprehensive report composed of PCR and Southern information, data and photos which indicate successful vector integration.
