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

Authors

  • Michael W. King,

    Corresponding author
    1. Center for Medical Education, Indiana University School of Medicine, Terre Haute, Indiana
    2. Indiana University Center for Regenerative Biology and Medicine, Indianapolis, Indiana
    • Terre Haute Center for Medical Education, Holmstedt Hall, Room 135, Indiana State University, Terre Haute, IN 47809
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  • Trent Nguyen,

    1. Medical Sciences Program, Indiana University School of Medicine, Bloomington, Indiana
    Current affiliation:
    1. Neurology Department, University of California, San Francisco VA Medical Center, San Francisco, California
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  • John Calley,

    1. Bio-Research Technologies & Proteins, Lilly Research Laboratories, Division of Eli Lilly & Company, Indianapolis, Indiana
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  • Mark W. Harty,

    1. Medical Sciences Program, Indiana University School of Medicine, Bloomington, Indiana
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  • Michael C. Muzinich,

    1. Medical Sciences Program, Indiana University School of Medicine, Bloomington, Indiana
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  • Anthony L. Mescher,

    1. Indiana University Center for Regenerative Biology and Medicine, Indianapolis, Indiana
    2. Medical Sciences Program, Indiana University School of Medicine, Bloomington, Indiana
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  • Chris Chalfant,

    1. Bio-Research Technologies & Proteins, Lilly Research Laboratories, Division of Eli Lilly & Company, Indianapolis, Indiana
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  • Mathias N'Cho,

    1. Bio-Research Technologies & Proteins, Lilly Research Laboratories, Division of Eli Lilly & Company, Indianapolis, Indiana
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  • Kevin McLeaster,

    1. Bio-Research Technologies & Proteins, Lilly Research Laboratories, Division of Eli Lilly & Company, Indianapolis, Indiana
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  • Jacquelyn McEntire,

    1. Bio-Research Technologies & Proteins, Lilly Research Laboratories, Division of Eli Lilly & Company, Indianapolis, Indiana
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  • David Stocum,

    1. Indiana University Center for Regenerative Biology and Medicine, Indianapolis, Indiana
    2. Department of Biology, Indiana University Purdue University Indianapolis, Indianapolis, Indiana
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  • Rosamund C. Smith,

    1. Indiana University Center for Regenerative Biology and Medicine, Indianapolis, Indiana
    2. Bio-Research Technologies & Proteins, Lilly Research Laboratories, Division of Eli Lilly & Company, Indianapolis, Indiana
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  • Anton W. Neff

    1. Indiana University Center for Regenerative Biology and Medicine, Indianapolis, Indiana
    2. Medical Sciences Program, Indiana University School of Medicine, Bloomington, Indiana
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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. Developmental Dynamics 226:398–409, 2003. © 2003 Wiley-Liss, Inc.

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