Identification of genes expressed during Xenopus laevis limb regeneration by using subtractive hybridization

Michael W. King, Trent Nguyen, John Calley, Mark W. Harty, Michael C. Muzinich, Anthony L. Mescher, Chris Chalfant, Mathias N'Cho, Kevin McLeaster, Jacquelyn McEntire, David Stocum, Rosamund C. Smith, Anton W. Neff

Research output: Contribution to journalArticle

40 Scopus citations

Abstract

Suppression polymerase chain reaction-based subtractive hybridization was used to identify genes that are expressed during Xenopus laevis hindlimb regeneration. Subtractions were done by using RNAs extracted from the regeneration-competent stage (stage 53) and regeneration-incompetent stage (stage 59) of limb development. Forward and reverse subtractions were done between stage 53 7-day blastema and stage 53 contralateral limb (competent stage), stage 59 7-day pseudoblastema and stage 59 contralateral limb (incompetent stage), and stage 53 7-day blastema and stage 59 7-day pseudoblastema. Several thousand clones were analyzed from the various subtracted libraries, either by random selection and sequencing (1,920) or by screening subtracted cDNA clones (6,150), arrayed on nylon membranes, with tissue-specific probes. Several hundred clones were identified from the array screens whose expression levels were at least twofold higher in experimental tissue vs. control tissue (e.g., blastema vs. limb) and selected for sequencing. In addition, primers were designed to assay several of the randomly selected clones and used to assess the level of expression of these genes during regeneration and normal limb development. Approximately half of the selected clones were differentially expressed, as expected, including several that demonstrate blastema-specific enhancement of expression. Three distinct categories of expression were identified in our screens: (1) clones that are expressed in both regeneration-competent blastemas and -incompetent pseudoblastemas, (2) clones that are expressed at highest levels in regeneration-competent blastemas, and (3) clones that are expressed at highest levels in regeneration-incompetent pseudoblastemas. Characterizing the role of each of these three categories of genes will be important in furthering our understanding of the process of tissue regeneration.

Original languageEnglish (US)
Pages (from-to)398-409
Number of pages12
JournalDevelopmental Dynamics
Volume226
Issue number2
DOIs
StatePublished - Feb 1 2003

Keywords

  • Blastema
  • cDNA
  • Regeneration
  • Subtractive hybridization
  • Xenopus

ASJC Scopus subject areas

  • Developmental Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Identification of genes expressed during Xenopus laevis limb regeneration by using subtractive hybridization'. Together they form a unique fingerprint.

  • Cite this

    King, M. W., Nguyen, T., Calley, J., Harty, M. W., Muzinich, M. C., Mescher, A. L., Chalfant, C., N'Cho, M., McLeaster, K., McEntire, J., Stocum, D., Smith, R. C., & Neff, A. W. (2003). Identification of genes expressed during Xenopus laevis limb regeneration by using subtractive hybridization. Developmental Dynamics, 226(2), 398-409. https://doi.org/10.1002/dvdy.10250