However, using homologous recombination in ES cells to generate mutant mice is an inefficient, slow and expensive method because (i) homologous recombination typically occurs at low frequency in ES cells, requiring sophisticated selection schemes and screening of hundreds of clones to identify the desired mutant; (ii) large (15–20 kb) plasmids, often difficult to clone, are required to increase targeting efficiency and (iii) breeding mice to homozygosity and housing a mouse colony generate further delays and costs. Such limitations make the repeated targeting of a locus a technically daunting and economically impractical task.