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Artificial chromosome transgenesis reveals long-distance negative regulation of rag1 in zebrafish

Nature Genetics volume 23, pages 15–16 (1999)Cite this article

The ability of the immune system to respond to an endless number of foreign antigens is conferred by V(D)J recombination1, which occurs specifically in immature lymphocytes and is controlled by the highly conserved recombination activating genes rag1 and rag2 (refs 2–4). Despite the essential roles of these genes, the mechanisms responsible for restricting their expression to lymphoid cells are undefined5,6,7,8. Using artificial chromosome transgenesis in zebrafish, we have found evidence that distal negative regulatory elements are critical to silence inappropriate rag1 expression and have identified the olfactory tissues as a novel rag expression site in zebrafish.

These results suggested that our reporter constructs lacked a regulatory element necessary to suppress rag1 expression in non-lymphoid tissues. Therefore, we expanded the region of the rag1 locus under analysis using a 125-kb P1-derived artificial chromosome (PAC) that included 80 kb of 5´ and 40 kb of 3´ sequences flanking rag1. Chi-stimulated homologous recombination11 was used to insert the gene encoding GFP into this PAC (Fig. 1c). In contrast to smaller constructs, the rag PAC mediated high GFP expression in a pattern reflective of endogenous rag1 (Fig. 1g,h), indicating that at least one negative regulatory element is required to eliminate inappropriate rag1 expression. Using the modified rag PAC as a source for additional reporter constructs, we have mapped the location of a regulatory element that suppresses GFP expression in the skeletal muscle of transgenic zebrafish to 8.1–13.5 kb 5´ of rag1 (Fig. 1i). Other negative regulatory elements are required to further restrict rag1 expression to the appropriate tissues.

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Figure 1: Distal negative regulatory elements confer tissue-specific expression of zebrafish rag1.
Figure 2: Zebrafish rag1 is expressed in olfactory tissues.

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Acknowledgements

We thank members of our laboratories for discussions and assistance, and L. Steiner for helpful discussions and support. This work was supported by grants from NIH to S.L. (R01 RR13227-01) and L. Steiner (2RO1 AI08054). C.E.W. supported by NIH grant 9T32 AI07463.

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Authors and Affiliations

  1. Institute of Molecular Medicine and Genetics and Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, 30912, Georgia, USA

    Jason R. Jessen & Shuo Lin

  2. Department of Biology, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts, USA

    Catherine E. Willett

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  1. Jason R. Jessen
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Correspondence to Shuo Lin.

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Jessen, J., Willett, C. & Lin, S. Artificial chromosome transgenesis reveals long-distance negative regulation of rag1 in zebrafish. Nat Genet 23, 15–16 (1999). https://doi.org/10.1038/12609

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