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A Simple Guide for Generating BAC Transgenic Animals for Retinal Research.

Cavit Agca1,2,3, Christian Grimm4,5,6

  • 1Lab for Retinal Cell Biology, Department of Ophthalmology, University of Zurich, Zurich, Switzerland. cavit.agca@sabanciuniv.edu.

Methods in Molecular Biology (Clifton, N.J.)
|December 2, 2019
PubMed
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Bacterial artificial chromosomes (BACs) enable the study of gene regulation by carrying large DNA segments. This study details a simplified BAC transgenesis method using galK-based recombineering to create a BAC:LIF-EGFP mouse model.

Area of Science:

  • Genomics
  • Molecular Biology
  • Transgenic Technology

Background:

  • Bacterial artificial chromosomes (BACs) are large DNA constructs used in genomics.
  • BACs can accommodate hundreds of kilobases of exogenous DNA, encompassing regulatory elements.
  • Analyzing gene regulation requires tools that preserve endogenous cis-regulatory elements.

Purpose of the Study:

  • To illustrate a simplified method for BAC transgenesis.
  • To generate a BAC:LIF-EGFP transgenic mouse line for studying gene expression.
  • To demonstrate the utility of galK-based recombineering in BAC construction.

Main Methods:

  • Generation of a Bacterial Artificial Chromosome (BAC) construct.
  • Utilizing galK-based recombineering for simplified BAC transgenesis.
Keywords:
BAC sequencingBAC transgenicsColony real-time PCRElectroporationLifMinimal mediaRecombineeringSW102galK

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  • Creation of a BAC:LIF-EGFP transgenic mouse line.

    • Main Results:

    • Successful generation of a BAC:LIF-EGFP transgenic mouse line.
    • Demonstration of a simplified BAC transgenesis protocol.
    • Validation of BACs for studying endogenous gene regulation in vivo.

    Conclusions:

    • BAC transgenesis is a powerful tool for analyzing gene regulation.
    • GalK-based recombineering offers a simplified approach to BAC vector construction.
    • The BAC:LIF-EGFP mouse model serves as a valuable resource for gene expression studies.