Gorbsky GJ et al. (2022), Developing immortal cell lines from Xenopus emb...

XB-ART-59218

Open Biol 2022 Jul 01;127:220089. doi: 10.1098/rsob.220089.

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Developing immortal cell lines from Xenopus embryos, four novel cell lines derived from Xenopus tropicalis.

Gorbsky GJ , Daum JR , Sapkota H , Summala K , Yoshida H , Georgescu C , Wren JD , Peshkin L , Horb ME .

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The diploid anuran Xenopus tropicalis has emerged as a key research model in cell and developmental biology. To enhance the usefulness of this species, we developed methods for generating immortal cell lines from Nigerian strain (NXR_1018, RRID:SCR_013731) X. tropicalis embryos. We generated 14 cell lines that were propagated for several months. We selected four morphologically distinct lines, XTN-6, XTN-8, XTN-10 and XTN-12 for further characterization. Karyotype analysis revealed that three of the lines, XTN-8, XTN-10 and XTN-12 were primarily diploid. XTN-6 cultures showed a consistent mixed population of diploid cells, cells with chromosome 8 trisomy, and cells containing a tetraploid content of chromosomes. The lines were propagated using conventional culture methods as adherent cultures at 30°C in a simple, diluted L-15 medium containing fetal bovine serum without use of a high CO2 incubator. Transcriptome analysis indicated that the four lines were distinct lineages. These methods will be useful in the generation of cell lines from normal and mutant strains of X. tropicalis as well as other species of Xenopus.

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Species referenced: Xenopus tropicalis

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	Figure 1. Phase-contrast images of four selected Xenopus tropicalis cell lines. Bar = 50 µm.
	Figure 2. Karyotype analysis of four selected X. tropicalis cell lines. (a) Quantitation of chromosome spreads from XTN-6, XTN-8, XTN-10 and XTN-12 cell lines. Karyotype categories are listed on the X axis and numbers of spreads of each category are listed above the bars. (b) Examples of normal diploid spreads (20 chromosomes) from each cell line. (c) Chromosome spreads from XTN-6 cells showing single examples of 21 chromosomes, single chromosome aneuploidy and 42 chromosomes, possibly a tetraploid cell derived from a cell which originally contained 21 chromosomes. (d) An example of a karyotype analysis which revealed that XTN-6 cells with 21 chromosomes contain an extra copy of chromosome 8. (Chromosomes are numbered according to the updated nomenclature [16]).
	Figure 3. Growth properties of four X. tropicalis cell lines.
	Figure 4. Expression of the senesce marker beta-galactosidase indicated by the blue colour in this phase-contrast image of XTN-12 cells initially plated at low density. Bar = 20 µm.
	Figure 5. Comparison of gene expression in XTN cell lines. (a) Four pair-wise comparisons of gene expression in the cell lines. The most highly expressed genes and outliers are labelled using a human symbol of the homologous gene. It is apparent that XTN-8 and XTN-10 are most similar to each other and XTN-6 is most different from the rest. (b) Dendrogram and expression comparing all four cell lines. The numerical scale on the dendrogram reflects similarity matrix using a cosine distance with blue indicating greater similarity and red greater dissimilarity between pairs of lines.
	Figure 6. Cells stably expressing EGFP-tubulin imaged with spinning disc confocal microscopy. (a) XTN-6 cell in mitosis. Image depicts cell at metaphase captured from a time lapse video recording which is shown in electronic supplementary material, video S1. (b) A large XTN-8 cell in interphase. Image depicts a flattened z projection of a live cell. Bars = 10 µm.
	Figure 7. XTN-12 cell expressing EGFP-tubulin imaged with light-sheet microscopy. (a) Initial frame from a video centered on the retracting lamella of a moving interphase cell. (b) Four instances of microtubule breaks occurring during the video sequence. The entire sequence is available as electronic supplementary material, video S3. Bar = 10 µm.

References [+] :

Anizet, Characterization of a new cell line, XL2, obtained from Xenopus laevis and determination of optimal culture conditions. 1981, Pubmed, Xenbase