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PubMed · 10407679

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

A C Boyd·H Davidson·B Stevenson·G McLachlan·H Davidson-Smith·D J Porteous
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Abstract

A modified bacterial artificial chromosome (BAC) vector, pSURF-2, adapted for the selective subcloning of yeast artificial chromosome (YAC) sequences was constructed. DH10B-U, a pyrF derivative of the highly transformable E. coli strain DH10B was also constructed and used for the detection of Ura+ recombinants 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 mouse tyrosinase minigene was cloned into pSURF-2. Appropriately spliced tyrosinase RNA was detected by reverse transcription (RT)-PCR in extracts of cells transiently lipofected with the cloned YAC. (ii) Cells expressing human cystic fibrosis transmembrane conductance regulator (CFTR) from an integrated pSURF-2 recombinant containing a cDNA expression cassette were selected using the hygromycin-resistance (HyTK) marker of the vector and characterized by RT-PCR and immunoprecipitation. The unique I-SceI site and HyTK marker of pSURF-2 are designed to facilitate subsequent functional studies of cloned DNA.

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AnimalsCell LineChromosomes, Artificial, YeastCinnamatesCloning, MolecularCystic Fibrosis Transmembrane Conductance RegulatorDNA, BacterialDNA, FungalDrug ResistanceElectrophoresis, Agar GelEscherichia coliGenetic MarkersGenetic VectorsHumansHygromycin BIntegrasesMiceMonophenol MonooxygenasePlasmidsReverse Transcriptase Polymerase Chain ReactionTransfectionViral ProteinsJournal ArticleResearch Support, Non-U.S. Gov't

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A C Boyd, H Davidson, B Stevenson, G McLachlan, H Davidson-Smith, D J Porteous. 1999. pSURF-2, a modified BAC vector for selective YAC cloning and functional analysis.. https://doi.org/10.2144/99271rr05

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