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Cell Rep. 2014 Jun 26;7(6):2078-86. doi: 10.1016/j.celrep.2014.05.031. Epub 2014 Jun 12.

A conditional system to specifically link disruption of protein-coding function with reporter expression in mice.

Author information

1
Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5120, USA.
2
David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
3
David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
4
Department of Cancer Biology, University of Pennsylvania, Philadelphia, PA 19104-6160, USA; Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA 19104-6160, USA.
5
Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5120, USA; Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305-5324, USA; Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA 94305-5456, USA. Electronic address: mwinslow@stanford.edu.

Abstract

Conditional gene deletion in mice has contributed immensely to our understanding of many biological and biomedical processes. Despite an increasing awareness of nonprotein-coding functional elements within protein-coding transcripts, current gene-targeting approaches typically involve simultaneous ablation of noncoding elements within targeted protein-coding genes. The potential for protein-coding genes to have additional noncoding functions necessitates the development of novel genetic tools capable of precisely interrogating individual functional elements. We present a strategy that couples Cre/loxP-mediated conditional gene disruption with faithful GFP reporter expression in mice in which Cre-mediated stable inversion of a splice acceptor-GFP-splice donor cassette concurrently disrupts protein production and creates a GFP fusion product. Importantly, cassette inversion maintains physiologic transcript structure, thereby ensuring proper microRNA-mediated regulation of the GFP reporter, as well as maintaining expression of nonprotein-coding elements. To test this potentially generalizable strategy, we generated and analyzed mice with this conditional knockin reporter targeted to the Hmga2 locus.

PMID:
24931605
PMCID:
PMC4113058
DOI:
10.1016/j.celrep.2014.05.031
[Indexed for MEDLINE]
Free PMC Article
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