3 NEW DEVELOPMENTS IN P1 CLONING - INCREASED CLONING EFFICIENCY, IMPROVED CLONE RECOVERY, AND A NEW P1 MOUSE LIBRARY

In this report, we describe three new P1 cloning developments. Two of these developments represent improvements in cloning efficiency and clone recovery, and the third is the production and partial characterization of a new P1 mouse library. To increase cloning efficiency, we have produced a new lysis-defective (Delta lydAB) P1 lysogen (NS3690) for the production of the stage II head-tail-P1 packaging extract that is easier to use than the original stage II lysogen (NS3210), and that produces stage II extracts that are five- to eightfold more efficient than the original extracts. We believe the increased efficiency is due to the more concentrated packaging components in the NS3690 extract. Regarding P1 clone recovery, we demonstrate here that the less than optimal recovery of P1 plasmid DNA from P1 clones is due to the continuous presence of the P1 Cre recombinase in the host strain containing those clones (NS3529). Consequently, a simple method of P1 plasmid clone transduction is described to transfer clone DNA from NS3529 (Cre(+)) to its Cre(-) parent (NS3516). Yields of P1 plasmid DNA from NS3516 are as much as tenfold higher than from NS3529. Finally, we document here the production of a new P1 mouse library that was generated using genomic DNA from embryonic stem cell line E14 (a 129/Ola mouse). The library contains 182,000 independent clones whose average insert size is 80 kb and, based on >100 polymerase chain reaction screens, has an average unique sequence-hit size of 4.6.