Relevance of BAC transgene copy number in mice: transgene copy number variation across multiple transgenic lines and correlations with transgene integrity and expression.
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.
Chandler
Kelly J
KJ
Department of Molecular Physiology and Biophysics, Center for Human Genetics Research, Vanderbilt University School of Medicine, 519 Light Hall, 2215 Garland Avenue, Nashville, Tennessee 37232, USA.
Chandler
Ronald L
RL
Broeckelmann
Eva M
EM
Hou
Yue
Y
Southard-Smith
E Michelle
EM
Mortlock
Douglas P
DP
D000818:Animals; D022202:Chromosomes, Artificial, Bacterial; D004247:DNA / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005821:Genetic Techniques; D051379:Mice; D008822:Mice, Transgenic; D008957:Models, Genetic; D009693:Nucleic Acid Hybridization; D010375:Pedigree; D011110:Polymorphism, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012685:Sepharose / Q000737:chemistry; D019076:Transgenes
D004247:DNA; D012685:Sepharose