Abstract
Bacterial artificial chromosomes (BACs) are excellent tools for manipulating large DNA fragments and, as a result, are increasingly utilized to engineer transgenic mice by pronuclear injection. The demand for BAC transgenic mice underscores the need for careful inspection of BAC integrity and fidelity following transgenesis, which may be crucial for interpreting transgene function. Thus, it is imperative that reliable methods for assessing these parameters are available. However, there are limited data regarding whether BAC transgenes routinely integrate in the mouse genome as intact molecules, how BAC transgenes behave as they are passed through the germline across successive generations, and how variation in BAC transgene copy number relates to transgene expression. To address these questions, we used TaqMan real-time PCR to estimate BAC transgene copy number in BAC transgenic embryos and lines. Here we demonstrate the reproducibility of copy number quantification with this method and describe the variation in copy number across independent transgenic lines. In addition, polymorphic marker analysis suggests that the majority of BAC transgenic lines contain intact molecules. Notably, all lines containing multiple BAC copies also contain all BAC-specific markers. Three of 23 founders analyzed contained BAC transgenes integrated into more than one genomic location. Finally, we show increased BAC transgene copy number correlates with increased BAC transgene expression. In sum, our efforts have provided a reliable method for assaying BAC transgene integrity and fidelity, and data that should be useful for researchers using BACs as transgenic vectors.
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Acknowledgments
The authors thank the Vanderbilt CHGR DNA Resources Core for real-time PCR assistance and TaqMan probe/primer design; Jeff Smith and Kevin Bradley for assistance in identification of STR markers within the Bmp4 BAC intervals; Jeff Innis and Maureen Gannon for helpful discussions on the manuscript; Nyk Reed for reading the manuscript; and Will Bush for statistical analysis and assistance. Kelly J. Chandler was supported by NIH Genetics Training Grant 1T32GM62758-03. Ronald L. Chandler was supported by NIH Developmental Biology Training Grant 5T32HD07502-08. Douglas P. Mortlock was supported by NIH Grants 1R01HD47880-01 and 5R01AR049529-04. Transgenic mice were generated by the Vanderbilt University VCSCV Transgenic Mouse and ES Cell Shared Resource, which is supported by the Vanderbilt Cancer, Diabetes, Kennedy, and Vision Centers. The authors acknowledge the use of the VUMC CHGR DNA Resources Core Facility.
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Chandler, K.J., Chandler, R.L., Broeckelmann, E.M. et al. Relevance of BAC transgene copy number in mice: transgene copy number variation across multiple transgenic lines and correlations with transgene integrity and expression. Mamm Genome 18, 693–708 (2007). https://doi.org/10.1007/s00335-007-9056-y
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DOI: https://doi.org/10.1007/s00335-007-9056-y