Deep screening of recombination proficient bacteriophage libraries.

We are using bacteriophage λ as an efficient vector for transferring genomic alterations to embryonic stem (ES) cells via gene targeting. We have developed a variety of phage-plasmid recombination techniques to direct mutations and modification cassettes to specific sites within phage targeting vectors (1,6–9). We have also made improvements on Seed’s original method of recombination screening of bacteriophage libraries (5), which allows phage clones to be isolated “genetically” using recombination. In retro-recombination screening (8), phage targeting vectors from an ES cell targeting vector library in λ thymidine kinase (TK) are purified on a restrictive host, following the homologous integration of a supFhomology-bearing plasmid, which suppresses Aam, Bam, and Sam mutations in suitable phage λ genes essential for phage growth. Because the region of homology is duplicated upon integration, and can therefore revert under relaxed conditions (i.e., within a supF host), we incorporated the gam gene into the recombination plasmid to select for phages that have reverted to their original configuration (8). Retro-recombination screening has several advantages over conventional phage library screening techniques. Specific targeting vector phages can be isolated without plaque hybridization and purified in 2–3 days to serve as templates for the rapid completion of targeting vector construction via recombination. Libraries are screened in culture tubes, meaning that multiple library screens (i.e., targeting vector homology region isolation) can be performed at one time and there is no shortage of the number of phages that can be screened simultaneously to ensure the isolation of distinct clones from the locus of interest. This latter point is particularly important in the case of our λTK library because the insert size is approximately 12 kb and as many as 2 × 106 phages need to be screened at one time to ensure sufficient representation of all genes within the library. One drawback of recombination screening approaches is that most libraries, including our λTK library, are constructed with commercial packaging extracts that are not irradiated when prepared. As a result, a small yet significant number of non-amber phages can appear in a library of amber mutationcontaining phages, which arise from recombination between the two mutant strains used to generate head and tail extracts. While the incidence of such phages is rare (i.e., 10-4), it presents a unique challenge in screening amplified libraries by recombination, as Benchmarks