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 cl one recovery, and the third is the production and partial characteriza tion of a new P1 mouse library. To increase cloning efficiency, we hav e 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 p roduces stage II extracts that are five- to eightfold more efficient t han the original extracts. We believe the increased efficiency is due to the more concentrated packaging components in the NS3690 extract. R egarding P1 clone recovery, we demonstrate here that the less than opt imal recovery of P1 plasmid DNA from P1 clones is due to the continuou s presence of the P1 Cre recombinase in the host strain containing tho se clones (NS3529). Consequently, a simple method of P1 plasmid clone transduction is described to transfer clone DNA from NS3529 (Cre(+)) t o 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 gen omic DNA from embryonic stem cell line E14 (a 129/Ola mouse). The libr ary contains 182,000 independent clones whose average insert size is 8 0 kb and, based on >100 polymerase chain reaction screens, has an aver age unique sequence-hit size of 4.6.