Molecular and Cellular Biochemistry

, Volume 373, Issue 1–2, pp 41–51 | Cite as

Development and characterization of Xl1, a Xenopus laevis chondrocyte-like cell culture

  • Natércia Conceição
  • Michael Viegas
  • João Fidalgo
  • M. Leonor Cancela
Article
  • 263 Downloads

Abstract

We describe the development and characterization of a new cell line, designated Xl1, derived from vertebra and long bones of Xenopus laevis. These cells can mineralize their extracellular matrix upon addition of an inorganic phosphate donor and vitamin C, as characterized by von Kossa staining. In addition they express genes such as matrix gla protein (mgp), alkaline phosphatase, type II collagen, and retinoic acid receptors, representing a valuable tool to analyze expression and regulation of Xenopus cartilage-associated genes. Continuous treatment with retinoic acid (RA) inhibited mineralization, alkaline phosphatase expression and its activity, suggesting that RA is a potential negative regulator of Xl1 cell differentiation. These cells are receptive to efficient transfer of DNA using conventional methods including calcium phosphate, liposome-mediated transfer or electroporation and were found to express basal levels of mgp at least 50-fold higher than the routinely used Xenopus A6 cell line, as seen by transcription assays with the distal X. laevis mgp promoter. Being the first amphibian cell line derived from bone tissue, the Xl1 culture provides an excellent in vitro tool for functional promoter studies, being suitable, among other uses, for identifying promoter elements mediating cartilage-expressed genes as shown here for mgp.

Keywords

Cell culture Chondrocyte Mineralization Gene expression Transfection 

Notes

Acknowledgments

This work was partially funded by CCMAR and grants POCI/BIABCM/58677/2004 (POCI 2010 and FEDER funding) from the Portuguese Science and Technology Foundation (FCT). NC is supported by a post-doctoral grant from FCT (SFRH/BPD/48206/2008). MV was funded by Instituto do Emprego e Formação Profissional (IEFP) and CCMAR (fellowship ref#72/EP/2008). We are grateful to Dr. Jorge Pinto for his help in the generation of the Xenopus Xl1 cells.

Supplementary material

11010_2012_1473_MOESM1_ESM.tif (116 kb)
Karyotype analysis of Xl1 cells. (A) Distribution of chromosome number based on 53 metaphase preparations. (B) Metaphase spread of Xl1 cells chromosomes (Giemsa stain) (TIFF 115 kb)
11010_2012_1473_MOESM2_ESM.tif (282 kb)
Effect of culture conditions on mineralization of Xl1 cells. Xl1 cells were left to differentiate under mineralized conditions in the presence or absence of warfarin treatment for 18 days and then stained by the von Kossa technique to show the presence of mineral associated with the matrix. Mineralization was increased with warfarin treatment. (Ct) normal growth medium, (MM-Ca) mineralizing medium without the CaCl2 supplement, (MM) normal mineralizing medium, (MM + Warf) mineralizing medium supplemented with 10 μg/mL warfarin (TIFF 282 kb)
11010_2012_1473_MOESM3_ESM.xls (24 kb)
Sequences of the pairs of oligonucleotides used for PCR analysis to amplify the corresponding Xenopus mRNAs (XLS 23 kb)

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Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Natércia Conceição
    • 1
  • Michael Viegas
    • 1
  • João Fidalgo
    • 1
    • 3
  • M. Leonor Cancela
    • 1
    • 2
  1. 1.Centre of Marine Sciences (CCMAR)University of AlgarveFaroPortugal
  2. 2.Department of Biomedical Sciences and MedicineUniversity of AlgarveFaroPortugal
  3. 3.Cytogenetics DepartmentSullivan Nicolaides PathologyBrisbaneAustralia

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