BAC Transgenes, DNA Purification, and Transgenic Mouse Production

  • Michael G. Zeidler
  • Margaret L. Van Keuren
  • Thomas L. Saunders
Protocol
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Transgenic mouse models open new avenues of research to understand gene function, to mark cells, and to model human genetic diseases. The use of large DNA transgenes provides more information to cells and tissues in the mouse so that gene expression occurs at physiological levels in the appropriate cell types while recapitulating normal developmental time frames. Genomic libraries prepared in bacterial artificial chromosomes (BACs) for the mouse and human genome sequencing projects are a ready source of large DNA transgenes. The use of recombineering technology to modify BAC clones for (1) expression of proteins resulting from point mutations, (2) marking of specific cell populations with fluorescent protein reporters, or (3) cell-specific expression of exogenous proteins that can be used to control inducible gene expression and enhance the utility of BAC transgene-based experimental models. Large BAC transgenes and small plasmid transgenes are used to produce transgenic founder mice by the microinjection of purified DNA into the pronuclei of fertilized mouse eggs in both cases. Despite this similarity, the application of small DNA injection methods to large DNA transgenes leads to the integration of DNA fragments instead of intact BAC transgenes. Important technical differences between small and large DNA transgenesis include differences in DNA purification, microinjection buffers, and genotyping strategies to detect genomic integration. Careful attention to detail results in rewarding mouse models that can be used to identify disease-causing mutations in spontaneous mouse mutants, to purify rare cells that express fluorescent markers, and to explore genetic elements that control gene expression.

Abbreviations

BAC

Bacterial Artificial Chromosome

bp

Base pair

C.H.O.R.I

Children’s Hospital Oakland Research Institute

NCBI

National Center for Biotechnology Information

NEB

New England Biolabs

PA

Polyamine

PFGE

Pulsed Field Gel Electrophoresis

pg

Picogram

SSAHA

Sequence Search and Alignment by Hashing Algorithm

UCSC

University of California Santa Cruz

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Copyright information

© Springer-Verlag Berlin Heidelberg 2011

Authors and Affiliations

  • Michael G. Zeidler
    • 1
  • Margaret L. Van Keuren
    • 2
  • Thomas L. Saunders
    • 3
    • 4
  1. 1.Transgenic Animal Model CoreUniversity of Michigan, Medical SchoolAnn ArborUSA
  2. 2.Transgenic Animal Model CoreUniversity of Michigan, Medical SchoolAnn ArborUSA
  3. 3.Transgenic Animal Model CoreUniversity of Michigan, Medical SchoolAnn ArborUSA
  4. 4.Division of Molecular Medicine and Genetics, Department of Internal MedicineUniversity of Michigan, Medical SchoolAnn ArborUSA

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