A vehicle for DNA transfer and for recovery of transferred genes: λ Charon phage-pBR322 hybrid

Abstract

Recombinant Charon 4A phages accommodating the Herpes simplex virus (HSV-1) thymidine kinase ( tk) gene, the ampicillin-resistance (Ap R) gene, and the replication origin of pBR322 were constructed. The phage DNA was introduced into mouse L tk − cells by a free DNA transfer method or phage-mediated DNA transfer method [Ishiura et al., Mol. Cell. Biol. 2 (1982) 607]. Analyses of the physical state of the transferred DNA in the recipient cell genome showed that a DNA fragment as long as 12.7 kb was integrated intact into 67% and less than 40% of the L tk − transformant cells by phage-mediated DNA transfer and by free DNA transfer, respectively. We also developed a new rapid method for recovery of the transferred gene from the L tk + cell into Escherichia coli; the method depends on the fact that the recombinant 2 phage carrying the Ap R gene and replication origin ofpBR322 transduces λ-lysogenic bacteria to Ap R and is maintained as a plasmid. Using this method the HSV-1 tk gene from one L tk + transformant was rapidly and successfully recovered without any rearrangement of the target sequence.