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Cell lines are useful tools to facilitate in vitro studies of many biological and molecular processes. We describe a new permanent fibroblast-type cell line obtained from disaggregated Xenopus tropicalis limb bud. The cell line population doubling time was ~24 h. Its karyotype was genetically stable with a chromosome number of 2n = 21 and a chromosome 10 trisomy. These cells could be readily transfected and expressed transgenes faithfully. We obtained stable transformants using transposon-based gene transfer technology. These cells responded to thyroid hormone and thus can provide a complementary research tool to study thyroid hormone signaling events. In conclusion, this cell line baptized "Speedy" should prove useful to couple in vitro and in vivo biological studies in the X. tropicalis frog model.
Figure 1. Biological and cytological characterization of the primary cell line 91.1.F1 and the secondary Speedy cell line originating from a X. tropicalis limb. (a) Chromosome assay on 91.1.F1 cells at passage 4. Histogramme color code is gray: 20 chromosomes per metaphase; black: 21 chromosomes; white: less than 20 or more than 21 chromosomes. (b) Morphology of Speedy cells in phase-contrast microscopy. (c) Chromosome assay on Speedy cells at passages 12 and 60. Histogram color code as in a. (d) Karyotype of Speedy cells.
Figure 2. Cytological localization of centromeric markers on Speedy chromosomes by FISH. (a) Probe names and corresponding genes (Khokha et al.,2009: Chr1/LG1 : mast3; Chr2/LG6 : epb41; Chr3/LG8 : gemin5; Chr4/LG7 : znf423; Chr5/LG9 : olig3; Chr6/LG2 : fbxl7; Chr7/LG4 : mat1a; Chr8/LG5 : naif1; Chr9/LG3 : stat4; Chr10/LG10 : ezh1. (b) Representative image of FISH analysis of chromosome 10 centromeric probe performed on Speedy cells. The white arrows show fluorescent signals at the centromeric area of all three chromosome 10.
Figure 3. Expression and cellular localization of fluorescent proteins in Speedy cells. (a) Transfectability of speedy cells at 48 h after transfection of pEGFP-C1 plasmid. Scale bar represent 600 μm. (b) Representative images of fluorescent cells transfected with the pEGFP-C1. (c) Representative images of fluorescent cells transfected with the pCS2-TdTomato-2A-GFP and the pCS2-TdTomato-m2A-GFP. (f, j) EGFP fluorescence; (g, k) DAPI staining; (h, l) TdTomato fluorescence; (i, m) Merge of EGFP, DAPI and TdTomato fluorescence. ⪠N ⫠indicates the number of cells observed from two independent transfection experiments. Scale bars represent 10 μm.
Figure 4. Transpositional activity of SB and PB transposon systems in Speedy cells. (a) Schematic representation of the mPB and SB100 transposon systems. The transposase-encoding plasmids (helper plasmids) expressing SB100 and mPB transposases are pCSB100XNpA and pCmPBNpA, respectively. The donor plasmids for SB100 and mPB transposon systems are pT2(SV40-neo) and pXLBacII(SV40-neo), respectively. (b) Transpositional activities of SB100 and mPB in Speedy cells. The transposition rate was calculated as the ratio between the numbers of resistant colonies obtained in the presence of transposase-expressing construct versus in the absence (N = 3 ± SEM). Various amount of helper plasmids, namely 1X and 10X representing 150 ng and 1.5 μg, respectively, were tested: (c) Sequence of integration sites of mPB and SB100 within Speedy gDNA. The TSDs specific to each transposon system (TA for SB100 and TTAA for mPB) are underlined. (d) Representative images of FISH experiments on PBcl6 cell line generated by mPB transposition. An interphase nucleus is represented on the left panel whereas a metaphasic spread is shown on the right panel. The white arrows show fluorescent signals.
Figure 5. Speedy cells response to TH treatment. The relative quantity of the rpl8, thra, thrb, and thbzip mRNA after a 7.5 h of treatment using 10 nM T3 is indicated. The values given are means of three measures. Normalization was performed based on odc transcript levels in treated versus untreated cells.
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