Enhanced Efficiency of pSV1-RecA-Based BAC Recombineering

The use of BAC clones to analyze the regulation of gene expression in transgenic mice has been hindered by the lack of powerful and reliable methods for their modification, compared to those available for YAC clones. Several groups have reported the development of methods for the modification of BAC clones by homologous recombination (3), and we have used the method described by Yang et al. (7) to introduce two reporter genes into BAC clones containing the Mrf4/Myf5 locus, which encodes two of the key transcription factors involved in skeletal muscle development. The targeted modification procedure developed by Yang et al. (7) is based on the pSV1-RecA shuttle vector that carries: (i) a tetracycline (TET) resistance gene for positive and negative selection; (ii) the RecA gene, which provides the enzymatic machinery needed for homologous recombination in E. coli; and (iii) a temperature-sensitive origin of replication that allows for the selection of vector DNA that has integrated into the BAC carried in the host cell. A modifying cassette containing 5′ and 3′ homology arms and the desired modification is subcloned into this vector, which is then used to transform BACcontaining E. coli. Growth on chloramphenicol (CAM) and TET plates at 30°C yields colonies that contain wildtype BAC that carry the CAM resistance gene and free shuttle vector. Homologous recombination results in the integration of the shuttle vector into the BAC clone, which gives rise to a co-integrate BAC and places shuttle vector sequences under the control of the BAC origin of replication. By shifting the temperature to 43°C, only cells containing co-integrate BAC clones should be TET-resistant. Growth at 43°C overnight of co-integrate BAC clones in the presence of CAM results in the formation of resolved BAC clones because of a second recombination event that removes the shuttle vector backbone sequences generating wild-type or modified BAC clones. Plating these colonies onto CAM/fusaric acid (FA) selects against TET-containing vectors; thus, only resolved BAC clones grow. However, shuttle vector replication is not completely suppressed at 43°C, and most of the colonies that grow after the temperature shift contain wild-type BAC and free shuttle vectors instead of co-integrate BAC clones. Where the locus to be modified shows low recombination frequencies, this method beBenchmarks