PSURF-2, a modified BAC vector for selective YAC cloning and functional analysis

A modified bacterial artificial chromosome (BAC) vector pSURF-2, adapted fo r the selective subcloning of yeast artificial chromosome (YAC) sequences w as constructed DH10B-U, a pyrF derivative of the highly transformable E. co li strain DH10B was also constructed and used for the detection of Ura(+) r ecombinants carrying DNA linked to YAC right arms. The vector's properties were illustrated in two main ways. (i) An intact 25-kb YAC containing a mou se tyrosinase minigene was cloned into pSURF-2. Appropriately spliced tyros inase RNA was detected by reverse transcription (RT)-PCR in extracts of cel ls transiently lipofected with the cloned YAC. (ii) Cells expressing human cystic fibrosis transmembrane conductance regulator (CFTR) from an integrat ed pSURF-2 recombinant containing a cDNA expression cassette were selected using the hygromycin-resistance (HyTK) marker of the vector and characteriz ed by RT-PCR and immunoprecipitation. The unique I-SceI site and HyTK marke r of pSURF-2 are designed to facilitate subsequent functional studies of cl oned DNA.