title: Regulatory elements of Xenopus col2a1 drive cartilaginous gene expression in transgenic frogs.
abstract: This study characterizes regulatory elements of collagen 2 alpha 1 (col2a1) in Xenopus that enable transgene expression in cartilage-forming chondrocytes. The reporters described in this study drive strong cartilage-specific gene expression, which will be a valuable tool for further investigations of Xenopus skeletal development. While endogenous col2a1 mRNA is expressed in many embryonic tissues, its expression becomes restricted to tadpole and adult chondrocytes. This chondrocyte-specific expression is recapitulated by col2a1 reporter constructs, which were tested through I-SceI meganuclease-mediated transgenesis. These constructs contain a portion of the Xenopus tropicalis col2a1 intron, which aligns to a cartilage-specific intronic enhancer that has been well characterized in mammals. Two overlapping regions of the first intron that are 1.5-Kb and 665-bp long, both of which contain this enhancer sequence, drove EGFP expression in both larval and adult chondrocytes when connected to an upstream promoter. However, neither a truncated 155-bp region that also contains the enhancer, nor a separate 347-bp intronic region that lacks it, was able to drive cartilaginous transgene expression. The two cartilage-specific transgenes are heritable in F1 progeny, which exhibit none of the background expression observed in the injected founders. This study is the first to use the I-SceI technique to characterize an enhancer element in Xenopus, and the first to generate chondrocyte-specific gene expression in a non-mammalian vertebrate. The creation of novel cartilage-specific gene expression provides a new tool for further studies of anuran skeletal development.
authors.affiliation: Biology Department, Dalhousie University, Halifax, NS, Canada. ryankerney@gmail.com
authors.forename: Ryan
authors.initials: R
authors.lastname: Kerney
authors.forename: Brian K
authors.initials: BK
authors.lastname: Hall
authors.forename: James
authors.initials: J
authors.lastname: Hanken
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D015415:Biomarkers / Q000378:metabolism; D002356:Cartilage / Q000166:cytology / Q000502:physiology*; D019902:Chondrocytes / Q000378:metabolism; D024043:Collagen Type II / Q000235:genetics* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D017403:In Situ Hybridization; D015347:RNA Probes; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D019076:Transgenes / Q000502:physiology*; D014982:Xenopus laevis / Q000196:embryology*
chemical_list: D015415:Biomarkers; D024043:Collagen Type II; D015347:RNA Probes; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C066166:AI4 protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific