title: A conditional system to specifically link disruption of protein-coding function with reporter expression in mice.
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.
authors.affiliation: Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5120, USA.
authors.forename: Shin-Heng
authors.initials: SH
authors.lastname: Chiou
authors.affiliation: David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Caroline
authors.initials: C
authors.lastname: Kim-Kiselak
authors.affiliation: Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5120, USA.
authors.forename: Viviana I
authors.initials: VI
authors.lastname: Risca
authors.affiliation: David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Megan K
authors.initials: MK
authors.lastname: Heimann
authors.affiliation: Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5120, USA.
authors.forename: Chen-Hua
authors.initials: CH
authors.lastname: Chuang
authors.affiliation: David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Aurora A
authors.initials: AA
authors.lastname: Burds
authors.affiliation: Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5120, USA.
authors.forename: William J
authors.initials: WJ
authors.lastname: Greenleaf
authors.affiliation: 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.
authors.forename: Tyler E
authors.initials: TE
authors.lastname: Jacks
authors.affiliation: 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.
authors.forename: David M
authors.initials: DM
authors.lastname: Feldser
authors.affiliation: 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.
authors.forename: Monte M
authors.initials: MM
authors.lastname: Winslow
mesh_terms: D000818:Animals; D005260:Female; D018390:Gene Targeting / Q000379:methods*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics*; D008297:Male; D051379:Mice; D011995:Recombination, Genetic
chemical_list: D049452:Green Fluorescent Proteins