Generating transgenic mice from bacterial artificial chromosomes: transgenesis efficiency, integration and expression outcomes |
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Authors: | Margaret L Van Keuren Galina B Gavrilina Wanda E Filipiak Michael G Zeidler Thomas L Saunders |
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Institution: | (1) Transgenic Animal Model Core, University of Michigan Medical School, 2560 MSRB II, 1150 West Medical Center Drive, SPC 5674, Ann Arbor, MI 48109, USA;(2) Department of Internal Medicine, Division of Molecular Medicine and Genetics, University of Michigan Medical School, Ann Arbor, MI 48109, USA |
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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. |
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Keywords: | Mice Transgenic Gene transfer techniques Chromosomes Artificial Bacterial BAC Electrophoresis Gel Pulsed-field DNA Gene Expression |
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