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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.

Regulatory elements of Xenopus col2a1 drive cartilaginous gene expression in transgenic frogs