Screening homologous recombination in ES cells
To screen and confirm homologous recombination (HR) in Embryonic Stem (ES) cells, extracted DNA from individual ES cells is first genotyped using a Loss-of-Allele (LOA) assay. LOA is a quantitative TaqMan® qPCR detecting the loss of one region of the native target due to correct homologous recombination with the gene targeting vector. In HR positive clones, we expect to detect one copy of this region in the recombined allele and two copies in the non-recombined, or wild type, allele. Assays are developed and validated before samples are processed, in triplicate, on an automated QuantStudio7 in 384 well plates. Samples are then analyzed by relative cycle threshold method. Once the initial candidates are identified and further expanded from 96 well plates to 6 well plates in the ES cell laboratory, the selected clones are then confirmed with LOA, selection cassette copy number analysis (ensuring there is only one copy of the selection cassette and not a targeted as well as random integrant), LoxP check for conditional alleles (ensuring both LoxP sites remained intact during homologous recombination) and Long Range PCR across both 5’ and 3’ arms of homology (PCR from vector/non genomic DNA to outside of the long arm of homology). In addition, we also offer high throughput screening to detect successful FLP/Cre recombinase mediated excision of the selection cassette(s).