Transgenic Research

, Volume 18, Issue 5, pp 769–785 | Cite as

Generating transgenic mice from bacterial artificial chromosomes: transgenesis efficiency, integration and expression outcomes

  • Margaret L. Van Keuren
  • Galina B. Gavrilina
  • Wanda E. Filipiak
  • Michael G. Zeidler
  • Thomas L. Saunders
Original Paper

Abstract

Transgenic mice are widely used in biomedical research to study gene expression, developmental biology, and gene therapy models. Bacterial artificial chromosome (BAC) transgenes direct gene expression at physiological levels with the same developmental timing and expression patterns as endogenous genes in transgenic animal models. We generated 707 transgenic founders from 86 BAC transgenes purified by three different methods. Transgenesis efficiency was the same for all BAC DNA purification methods. Polyamine microinjection buffer was essential for successful integration of intact BAC transgenes. There was no correlation between BAC size and transgenic rate, birth rate, or transgenic efficiency. A narrow DNA concentration range generated the best transgenic efficiency. High DNA concentrations reduced birth rates while very low concentrations resulted in higher birth rates and lower transgenic efficiency. Founders with complete BAC integrations were observed in all 47 BACs for which multiple markers were tested. Additional founders with BAC fragment integrations were observed for 65% of these BACs. Expression data was available for 79 BAC transgenes and expression was observed in transgenic founders from 63 BACs (80%). Consistent and reproducible success in BAC transgenesis required the combination of careful DNA purification, the use of polyamine buffer, and sensitive genotyping assays.

Keywords

Mice, Transgenic Gene transfer techniques Chromosomes, Artificial, Bacterial BAC Electrophoresis, Gel, Pulsed-field DNA Gene Expression 

Notes

Acknowledgments

We thank Tina Jones and Corey Ziebell for their management of the transgenic production mouse colonies and we thank Susan Allen for her editorial assistance. The Transgenic Animal Model Core of the University of Michigan’s Biomedical Research Core Facilities is supported by the University of Michigan Cancer Center (NIH CA046592), the University of Michigan Rheumatic Diseases Center Core (NIH AR048310), the University of Michigan Gastrointestinal Peptide Research Center (NIH DK034933), and the Nathan Shock Center for the Biology of Aging (NIH AG013283).

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Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Margaret L. Van Keuren
    • 1
  • Galina B. Gavrilina
    • 1
  • Wanda E. Filipiak
    • 1
  • Michael G. Zeidler
    • 1
  • Thomas L. Saunders
    • 1
    • 2
  1. 1.Transgenic Animal Model CoreUniversity of Michigan Medical SchoolAnn ArborUSA
  2. 2.Department of Internal Medicine, Division of Molecular Medicine and GeneticsUniversity of Michigan Medical SchoolAnn ArborUSA

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