Gene Arrays from Subtracted Libraries to Identify Regulators of Regeneration in Xenopus
Michael W. King1*, Anthony L. Mescher2*, Rosamund C. Smith3*, Anton W. Neff3*
Suppression PCR-based subtractive hybridization was used to identify genes that are expressed during Xenopus laevis hindlimb regeneration. Subtractions were done using RNAs extracted from limbs and regeneration blastemas at the regeneration-competent stage (stage 53) and regeneration-incompetent stage (stage 59) of development. Forward and reverse subtractions were done between stage 53 7-day blastema and stage 53 contralateral limb, stage 59 7-day pseudoblastema and stage 59 contralateral limb, 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 (1920) or by screening subtracted cDNA clones (6150), arrayed on nylon membranes, with tissue-specific probes. Several hundred clones were identified from the array screens whose expression levels were at least two-fold higher in experimental tissue versus control tissue (e.g. blastema versus 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 one 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.
1Indiana University School of Medicine, Terre Haute Center for Medical Education, Terre Haute, IN. 47809
2Indiana University School of Medicine, Medical Sciences Program, Bloomington, IN. 47405
3Bio-Research Technologies & Proteins, Eli Lilly & Company, Indianapolis, IN. 46206
*Indiana University Center for Regenerative Biology and Medicine