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Related Experiment Videos

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

A C Boyd1, H Davidson, B Stevenson

  • 1MRC Human Genetics Unit, Edinburgh, Scotland, UK. chrisb@hgu.mrc.ac.uk

Biotechniques
|July 17, 1999
PubMed
Summary
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A new bacterial artificial chromosome (BAC) vector, pSURF-2, enables selective subcloning of yeast artificial chromosome (YAC) DNA. This tool facilitates functional studies of cloned genes, demonstrated with mouse tyrosinase and human CFTR expression.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs) are crucial for cloning large DNA fragments.
  • Efficient methods for manipulating and studying YAC-derived sequences within BAC vectors are needed for functional genomics.
  • The development of specialized vectors can streamline the process of gene cloning and functional analysis.

Purpose of the Study:

  • To construct and characterize a modified BAC vector, pSURF-2, for the selective subcloning of YAC sequences.
  • To demonstrate the utility of pSURF-2 in facilitating functional studies of cloned DNA, including gene expression.
  • To develop a system for detecting recombinants carrying YAC DNA linked to specific genetic markers.

Main Methods:

Related Experiment Videos

  • Construction of a modified BAC vector (pSURF-2) and a specialized E. coli strain (DH10B-U).
  • Cloning of a YAC containing a mouse tyrosinase minigene into pSURF-2.
  • Transient lipofection and analysis of gene expression using reverse transcription PCR (RT-PCR).
  • Selection and characterization of cells expressing human cystic fibrosis transmembrane conductance regulator (CFTR) using a hygromycin-resistance marker and RT-PCR/immunoprecipitation.

    • Main Results:

    • The pSURF-2 vector successfully facilitated the cloning of a YAC containing a mouse tyrosinase minigene.
    • Functional expression of the mouse tyrosinase minigene was confirmed by detecting spliced RNA via RT-PCR.
    • The vector's hygromycin-resistance marker enabled selection of cells expressing human CFTR, with expression validated by RT-PCR and immunoprecipitation.
    • The I-SceI site and HyTK marker facilitate subsequent functional studies of cloned DNA.

    Conclusions:

    • The pSURF-2 BAC vector is a valuable tool for the selective subcloning and functional analysis of YAC DNA.
    • The vector system aids in the study of gene function by enabling expression and selection of cloned sequences.
    • pSURF-2 simplifies the process of preparing YAC inserts for downstream functional genomics applications.