Roy S, Gardiner D M, Bryant S V
Developmental Biology Center and Department of Developmental and Cell Biology, University of California at Irvine, Irvine, California 92697, USA.
Dev Biol. 2000 Feb 15;218(2):199-205. doi: 10.1006/dbio.1999.9556.
Axolotls, with their extensive abilities to regenerate as adults, provide a useful model in which to study the mechanisms of regeneration in a vertebrate, in hopes of understanding why other vertebrates cannot regenerate. Although the expression of many genes has been described in regeneration, techniques for functional analysis have so far been limited. In this paper we demonstrate a new method for efficient overexpression of foreign genes in axolotls. Using vaccinia virus expressing beta-galactosidase microinjected into regenerating limbs, we show that vaccinia can infect both dividing and nondividing limb cells. The site of infection remains discrete and there is no secondary spread of infection to nearby cells. beta-Gal is expressed at high levels in blastema cells for about a week and in differentiated cells for longer. Blastemas that have been injected with vaccinia at different stages regenerate normally. As a test of the utility of vaccinia for functional analysis in regeneration, we constructed a virus expressing Shh and injected it into the anterior of regenerating limbs. Ectopic Shh expression caused extra digits, carpals, and tarsals in the hands and feet of regenerating limbs, suggesting that despite differences in the timing of expression and the eventual pattern, the function of Shh appears to be similar to that in the developing limbs of other vertebrates. Our results demonstrate that vaccinia virus is an excellent vector for ectopically expressing genes for secreted proteins and is a useful tool to study the function of signaling molecules during the process of regeneration in urodeles.
美西螈作为成年个体具有广泛的再生能力，为研究脊椎动物的再生机制提供了一个有用的模型，以期了解其他脊椎动物不能再生的原因。尽管在再生过程中已经描述了许多基因的表达，但迄今为止，功能分析技术仍然有限。在本文中，我们展示了一种在美西螈中高效过表达外源基因的新方法。通过将表达β-半乳糖苷酶的痘苗病毒显微注射到正在再生的肢体中，我们发现痘苗病毒能够感染正在分裂和未分裂的肢体细胞。感染部位保持离散，且不会向附近细胞发生继发性感染扩散。β-半乳糖苷酶在芽基细胞中高水平表达约一周，在分化细胞中表达时间更长。在不同阶段注射痘苗病毒的芽基正常再生。作为对痘苗病毒在再生功能分析中的效用的测试，我们构建了一种表达Shh的病毒并将其注射到正在再生的肢体前部。异位表达的Shh导致正在再生的肢体的手和脚出现额外的指、腕骨和跗骨，这表明尽管表达时间和最终模式存在差异，但Shh的功能似乎与其他脊椎动物发育中的肢体相似。我们的结果表明，痘苗病毒是一种用于异位表达分泌蛋白基因的优秀载体，并且是研究有尾两栖类动物再生过程中信号分子功能的有用工具。
