Advanced Protocols for Animal Transgenesis

Part of the series Springer Protocols Handbooks pp 159-179


BAC Transgenes, DNA Purification, and Transgenic Mouse Production

  • Michael G. ZeidlerAffiliated withTransgenic Animal Model Core, University of Michigan, Medical School Email author 
  • , Margaret L. Van KeurenAffiliated withTransgenic Animal Model Core, University of Michigan, Medical School
  • , Thomas L. SaundersAffiliated withTransgenic Animal Model Core, University of Michigan, Medical SchoolDivision of Molecular Medicine and Genetics, Department of Internal Medicine, University of Michigan, Medical School

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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.