Targeting Mutations to YACs by Homologous Recombination

There are innumerable instances when one wants to manipulate cloned DNA: point mutagenesis for analysis of protein function and transcription factor binding sites, introduction of insertions to produce fusion proteins or to introduce reporter genes or motifs, and creation of deletions to define functional regions of proteins. One of the advantages of cloning in yeast artificial chromosomes (YACs) is that the DNA is maintained in the yeast host where one can carry out sophisticated and subtle mutagenesis with ease. Simple manipulations, such as the insertion of a mammalian selectable marker into the arm of the YAC, can be carried out by a single step integration or replacement as described in Chapter 17 . However, two rounds of recombination can be used to make more subtle mutations such as point mutations.