Characterization of a novel type I keratin gene and generation of transgenic lines with fluorescent reporter genes driven by its promoter/enhancer in Xenopus laevis

Abstract

We investigated the characteristics of a novel type I keratin gene in Xenopus laevis during ontogenesis. The transcript was first detected in the posterior region at the late neurula stage, and then restricted to the fin and external gill during embryogenesis. To examine the transcriptional regulation of the keratin gene in vivo, we generated transgenic lines with fluorescent reporter genes driven by its 4.2-kb upstream sequence. The promoter/enhancer activity recapitulated the endogenous gene expression during embryogenesis. Sequential deletion analyses revealed that the regions proximal to the promoter were essential for fin-specific expression. Reporter expression was detected in various organs, including the fin and gill. In particular, robust expression was observed in the developing limbs and gill. The reporter fluorescence rapidly decreased with internal gill resorption during metamorphosis. The transgenic lines carrying the promoter/enhancer should represent valuable tools for elucidating the formation, development and resorption of various organs, especially the gill. Developmental Dynamics 239:3172-3181,

Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9

“…The EGFP gene was inserted just before the endogenous stop codon to express an fgk – EGFP fusion gene. Regarding fgk , transgenic X. laevis embryos have reportedly shown specific expression in the fin and the gill 23 . Consistent with this report, we obtained several embryos showing fluorescence specifically localized in the fin ( Fig.…”

Germline Transgenic Methods for Tracking Cells and Testing Gene Function during Regeneration in the Axolotl

“…To label other tissues important in regeneration, such as epidermis, muscle, and cartilage, we employed previously characterized Xenopus promoters driving EGFP, namely Keratin12:EGFP , CarAct:EGFP , and Col2α1:EGFP (Kerney et al., 2010; Ogino et al., 2006; Suzuki et al., 2010). The Keratin:EGFP animals express EGFP exclusively in the skin (Figures 1C, S1F, and S1G), whereas the CarAct:EGFP transgenics show robust expression in the myosin heavy-chain-expressing muscle and not in the PAX7-positive satellite cells, indicating faithful muscle-specific expression (Figures 1D, S1H, and S1I).…”

Spatiotemporal expression profile of no29/nucleophosmin3 in the intestine of Xenopus laevis during metamorphosis

“…Animals, sampling, and TH treatment X. laevis tadpoles were maintained and treated as previously described (Hasebe et al 2007(Hasebe et al , 2008Suzuki et al 2009Suzuki et al , 2010. Developmental stages (NF stage) were determined according to Nieuwkoop and Faber (1956).…”

“…The plasmid was amplified by PCR with SP6 and T3 promoter primers. cRNA sense and antisense probes were synthesized from the PCR product with SP6 or T7 RNA polymerase, respectively, by using digoxigenin RNA Labeling Mix (Suzuki et al 2010; Roche Applied Science, Indianapolis, Ind., USA). Intestinal fragments were isolated from the anterior part of the small intestine just after the bile duct junction from tadpoles at indicated stages and from T3-treated tadpoles.…”