Generation of target gene for knockout mice

Schematic of targeting vector and positive/negative selection. A schematic depicting a targeting vector is shown at the top. Plasmid backbone sequences are shown with thin lines. The herpes simplex virus-thymidine kinase (HSV-TK) box represents the negatively selected marker herpes virus thymidine kinase. The hatched boxes represent sequences present in the target gene. The NEOR box represents the positively selected marker neomycin resistance. When this targeting construct is transfected into (embryonic stem) ES cells, a small fraction of the transfected cells will undergo homologous recombination mediated by the target gene sequences included in the vector. This is shown schematically by the Xs representing crossover points. The location of the transcript of the target gene is shown by the heavy black arrow. A double recombination, as pictured, will substitute the neomycin resistance marker for the native portion of the gene lying between the crossover points. This deletion disrupts the gene, thereby "knocking out" its function. Positive selection is applied by growing the transfected cells in the presence of G418, a toxic drug that is inactivated by NEOR. Negative selection is applied by growing the transfected cells in the presence of gancyclovir, which is converted to a toxin by HSV-TK. Thus, the population is enriched for cells retaining NEOR and simultaneously losing HSV-TK.