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Strategies for Designing Transgenic DNA Constructs

  • Chengyu Liu
Part of the Methods in Molecular Biology book series (MIMB, volume 1027)

Abstract

Generation and characterization of transgenic mice are important elements of biomedical research. In recent years, transgenic technology has become more versatile and sophisticated, mainly because of the incorporation of recombinase-mediated conditional expression and targeted insertion, site-specific endonuclease-mediated genome editing, siRNA-mediated gene knockdown, various inducible gene expression systems, and fluorescent protein marking and tracking techniques. Site-specific recombinases (such as PhiC31) and engineered endonucleases (such as ZFN and Talen) have significantly enhanced our ability to target transgenes into specific genomic loci, but currently a great majority of transgenic mouse lines are continuingly being created using the conventional random insertion method. A major challenge for using this conventional method is that the genomic environment at the integration site has a substantial influence on the expression of the transgene. Although our understanding of such chromosomal position effects and our means to combat them are still primitive, adhering to some general guidelines can significantly increase the odds of successful transgene expression. This chapter first discusses the major problems associated with transgene expression, and then describes some of the principles for using plasmid and bacterial artificial chromosomes (BACs) for generating transgenic constructs. Finally, the strategies for conducting each of the major types of transgenic research are discussed, including gene overexpression, promoter characterization, cell-lineage tracing, mutant complementation, expression of double or multiple transgenes, siRNA knockdown, and conditional and inducible systems.

Key words

Transgene DNA construct Position effects BAC Plasmid Overexpression Insulator siRNA Recombinase Cre–loxP FLP-FRT Conditional Inducible Animal model 

Notes

Acknowledgments

This work was supported by the Intramural Research Program of the National Heart, Lung, and Blood Institute.

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Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Chengyu Liu
    • 1
  1. 1.iPSC and Genome Engineering CoreNational Heart, Lung, and Blood Institute, National Institutes of HealthBethesdaUSA

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