pSURF-2, a modified BAC vector for selective YAC cloning and functional analysis.
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.
MRC Human Genetics Unit, Edinburgh, Scotland, UK. chrisb@hgu.mrc.ac.uk
A C
AC
Boyd
H
H
Davidson
B
B
Stevenson
G
G
McLachlan
H
H
Davidson-Smith
D J
DJ
Porteous
D000818:Animals; D002460:Cell Line; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics*; D002934:Cinnamates; D003001:Cloning, Molecular; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics*; D005819:Genetic Markers / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000494:pharmacology; D019426:Integrases / Q000378:metabolism; D051379:Mice; D014442:Monophenol Monooxygenase / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014764:Viral Proteins
C505032:CFTR protein, human; D002934:Cinnamates; D004269:DNA, Bacterial; D004271:DNA, Fungal; D005819:Genetic Markers; D014764:Viral Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006921:Hygromycin B; C026273:hygromycin A; D014442:Monophenol Monooxygenase; C045073:Cre recombinase; D019426:Integrases