Di¡erential protein pro¢le in the ear-punched tissue of regeneration and non-regeneration strains of mice: a novel approach to explore the candidate genes for soft-tissue regeneration X. Li, S. Mohan, W. Gu, N. Miyakoshi, D.J. Baylink * Musculoskeletal Disease Center, JL Pettis VA Medical Center and Loma Linda University, 11201 Benton Street (151), Loma Linda, CA 92357, USA Received 29 March 2000; received in revised form 7 July 2000; accepted 14 July 2000 Abstract Wound repair/regeneration is a genetically controlled, complex process. In order to identify candidate genes regulating fast wound repair/ regeneration in soft-tissue, the temporal protein profile of the soft-tissue healing process was analyzed in the ear-punched tissue of regeneration strain MRL/MpJ-Fas lpr (MRL) mice and non-regeneration strain C57BL/6J(B6) mice using surface-enhanced laser desorption and ionization (SELDI) ProteinChip technology. Five candidate proteins were identified in which responses of MRL to the ear punch were 2^4-fold different compared to that of B6. Their corresponding genes were predicted using an antigen^antibody assay validated mass-based approach. Most of the predicted genes are known to play a role or are likely to play a role in the wound repair/regeneration. Of the five candidate proteins, the amount of the 23 560 Da protein in the ear-punched tissue was significantly correlated with the rate of ear healing in six representative strains of mice, making it a good candidate for fast wound repair/regeneration. We speculate that the increased concentration of the 23 560 Da protein in the wound tissue could stimulate the expression of various growth-promoting proteins and consequently speed up the wound repair/regeneration processes. Here, we have shown that examination of protein expression profile using SELDI technology, coupled with database search, is an alternative approach to search for candidate genes for wound repair/regeneration. This novel approach can be implemented in a variety of biological applications. ß 2000 Elsevier Science B.V. All rights reserved. Keywords : Wound repair; Regeneration; SELDI ProteinChip; Microarray; Protein pro¢le 1. Introduction It has been suggested that regeneration in amphibians is a fundamental property of vertebrate ancestors related to embryogenesis, which has been apparently lost in most vertebrate species [1^3]. Given the fact that urodeles have retained this ancestral property, it is possible that some of the mammalian species might retain the potential for tissue regeneration. The possibility has been strength- ened by a recent report that MRL/MpJ-Fas lpr (MRL) mouse, unlike all other strains of mice tested including C57BL/6 (B6), displays a remarkable capacity for rapid and complete ear wound closure that resembles regenera- tion [4]. The regeneration of ear tissue, which consists of several major cell types including chondrocyte, chondro- blasts, dermal cells, and ¢berblasts in MRL mice was in- dicated by the presence of normal dermal regrowth with organized extracellular matrix deposition, normal vascula- ture, and cartilage regrowth, and absence of scar forma- tion. In contrast, the 2-mm ear hole in B6 mice only healed about 50% with disorganized extracellular matrix deposition, obvious scar formation, and without cartilage regeneration. Further studies have shown that the healing capacity of MRL is a heritable quantitative trait and genes regulating the phenotype are mapped to ¢ve quantitative trait loci (QTL) regions [5]. The remarkable capacity for ear tissue regeneration of MRL mice makes them ideal models to study the molecular mechanisms that underlie tissue regeneration and to explore the potential of genet- ically manipulating regeneration in mammalian tissue that do not naturally regenerate. Many traditional methods such as di¡erential screening, di¡erential display, subtraction hybridization, sodium do- decyl sulfate^polyacrylamide gel electrophoresis (SDS^ PAGE), and two-dimensional gel electrophoresis have been used to study the molecular basis of the tissue regen- 0304-4165 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII:S0304-4165(00)00118-5 * Corresponding author. Fax: +1-909-796-1680; E-mail : david.baylink@med.va.gov Biochimica et Biophysica Acta 1524 (2000) 102^109 www.elsevier.com/locate/bba