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

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

Research output: Contribution to journal › Article › peer-review

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.

Original language	English
Pages (from-to)	164-70, 172, 175
Journal	Biotechniques
Volume	27
Issue number	1
Publication status	Published - 1999

Keywords / Materials (for Non-textual outputs)

Animals Cell Line Chromosomes, Artificial, Yeast/*genetics *Cinnamates Cloning, Molecular Cystic Fibrosis Transmembrane Conductance Regulator/genetics DNA, Bacterial/*genetics DNA, Fungal/genetics Drug Resistance/genetics Electrophoresis, Agar Gel Escherichia coli/*genetics Genetic Markers/genetics Genetic Vectors/*genetics Humans Hygromycin B/analogs & derivatives/pharmacology Integrases/metabolism Mice Monophenol Monooxygenase/genetics Plasmids/genetics Reverse Transcriptase Polymerase Chain Reaction Transfection *Viral Proteins

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title = "pSURF-2, a modified BAC vector for selective YAC cloning and functional analysis",

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.",

keywords = "Animals Cell Line Chromosomes, Artificial, Yeast/*genetics *Cinnamates Cloning, Molecular Cystic Fibrosis Transmembrane Conductance Regulator/genetics DNA, Bacterial/*genetics DNA, Fungal/genetics Drug Resistance/genetics Electrophoresis, Agar Gel Escherichia coli/*genetics Genetic Markers/genetics Genetic Vectors/*genetics Humans Hygromycin B/analogs \& derivatives/pharmacology Integrases/metabolism Mice Monophenol Monooxygenase/genetics Plasmids/genetics Reverse Transcriptase Polymerase Chain Reaction Transfection *Viral Proteins",

author = "Boyd, \{A. C.\} and H. Davidson and B. Stevenson and G. McLachlan and H. Davidson-Smith and Porteous, \{D. J.\}",

note = "Jul pSURF-2, a modified BAC vector for selective YAC cloning and functional analysis Journal Article Research Support, Non-U.S. Gov't United states eng",

year = "1999",

language = "English",

volume = "27",

pages = "164--70, 172, 175",

journal = "Biotechniques",

issn = "0736-6205",

publisher = "Eaton Publishing Company",

number = "1",

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TY - JOUR

T1 - pSURF-2, a modified BAC vector for selective YAC cloning and functional analysis

AU - Boyd, A. C.

AU - Davidson, H.

AU - Stevenson, B.

AU - McLachlan, G.

AU - Davidson-Smith, H.

AU - Porteous, D. J.

N1 - Jul pSURF-2, a modified BAC vector for selective YAC cloning and functional analysis Journal Article Research Support, Non-U.S. Gov't United states eng

PY - 1999

Y1 - 1999

N2 - 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.

AB - 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.

KW - Animals Cell Line Chromosomes, Artificial, Yeast/genetics Cinnamates Cloning, Molecular Cystic Fibrosis Transmembrane Conductance Regulator/genetics DNA, Bacterial/genetics DNA, Fungal/genetics Drug Resistance/genetics Electrophoresis, Agar Gel Escherichi

M3 - Article

SN - 0736-6205

VL - 27

SP - 164-70, 172, 175

JO - Biotechniques

JF - Biotechniques

IS - 1

ER -

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

Abstract

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