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High-throughput engineering of the mouse genome coupled with high-resolution expression analysis

A Corrigendum to this article was published on 01 July 2003

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Abstract

One of the most effective approaches for determining gene function involves engineering mice with mutations or deletions in endogenous genes of interest. Historically, this approach has been limited by the difficulty and time required to generate such mice. We describe the development of a high-throughput and largely automated process, termed VelociGene, that uses targeting vectors based on bacterial artificial chromosomes (BACs). VelociGene permits genetic alteration with nucleotide precision, is not limited by the size of desired deletions, does not depend on isogenicity or on positive–negative selection, and can precisely replace the gene of interest with a reporter that allows for high-resolution localization of target-gene expression. We describe custom genetic alterations for hundreds of genes, corresponding to about 0.5–1.0% of the entire genome. We also provide dozens of informative expression patterns involving cells in the nervous system, immune system, vasculature, skeleton, fat and other tissues.

*Note: In the author list of the AOP version of this article, the name of author Rostislav Chernomorsky was misspelled Rostislav Chernomorski. This has been corrected in the online and print versions of the article.

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Figure 1: Schematic representation of approach used to generate BAC-based targeting vectors (BACvecs).
Figure 2: Loss-of-native-allele assay.
Figure 3: Generation of complex alleles and large deletions, and targeting efficiencies.
Figure 4: High-throughput screening of reporter gene expression (lacZ reporter, in blue) in various VelociGene-generated mice (in whole-mount views, unless indicated otherwise).
Figure 5: Examples of genes expressed in similar locations with distinctive subdistributions (in whole-mount views, unless indicated otherwise).

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  • 30 May 2003

    replaced PDF with correct version (print was correct), ammended PDF with errata, footnote in SGML at abstract

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Acknowledgements

We thank Margaret Karow for intellectual input, Petra Kraus for analysis of gene expression patterns and Fred W. Alt, Craig Bassing, Richard Flavell, Mike Brown, Joe Goldstein, Eric Shooter and Kornelia Polyak for suggesting genes to target. We thank Amalia Dutra for advice and assistance with FISH. We would also like to thank Li Pan, Joyce McClain, Virginia Hughes, Jeffrey Vercollone, Kethi Mullei, Jorge Bermudez, Jennifer Leung, Ning Yuan, Samantha Park, Ruth Santos, Collen Correa, Huan Jiang, Jinsop Om, Youngli Chang, Haige Zhang and Jose Rodriguez for technical assistance, as well as Vicki Lan and Scott Staton for graphics support.

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Correspondence to George D Yancopoulos.

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The authors of this paper (except for A.F.S.) are employed at Regeneron Pharmaceuticals, Inc.

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Valenzuela, D., Murphy, A., Frendewey, D. et al. High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nat Biotechnol 21, 652–659 (2003). https://doi.org/10.1038/nbt822

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