Abuqarn Mehtap, Allmeling Christina, Amshoff Inga, Menger Bjoern, Nasser Inas, Vogt Peter M, Reimers Kerstin
Departmen tof Plastic, Hand and Reconstructive Surgery, Medical School of Hannover, Podbielskistrasse 380, D-30659 Hannover, Germany.
Biochim Biophys Acta. 2011 Jul;1814(7):843-9. doi: 10.1016/j.bbapap.2011.04.018. Epub 2011 May 6.
Urodele amphibians are exceptional in their ability to regenerate complex body structures such as limbs. Limb regeneration depends on a process called dedifferentiation. Under an inductive wound epidermis terminally differentiated cells transform to pluripotent progenitor cells that coordinately proliferate and eventually redifferentiate to form the new appendage. Recent studies have developed molecular models integrating a set of genes that might have important functions in the control of regenerative cellular plasticity. Among them is Msx1, which induced dedifferentiation in mammalian myotubes in vitro. Herein, we screened for interaction partners of axolotl Msx1 using a yeast two hybrid system. A two hybrid cDNA library of 5-day-old wound epidermis and underlying tissue containing more than 2×10⁶ cDNAs was constructed and used in the screen. 34 resulting cDNA clones were isolated and sequenced. We then compared sequences of the isolated clones to annotated EST contigs of the Salamander EST database (BLASTn) to identify presumptive orthologs. We subsequently searched all no-hit clone sequences against non redundant NCBI sequence databases using BLASTx. It is the first time, that the yeast two hybrid system was adapted to the axolotl animal model and successfully used in a screen for proteins interacting with Msx1 in the context of amphibian limb regeneration.
有尾两栖动物在再生复杂身体结构（如四肢）方面具有非凡的能力。肢体再生依赖于一个称为去分化的过程。在诱导性伤口表皮下，终末分化细胞转变为多能祖细胞，这些祖细胞协调增殖并最终重新分化形成新的附属肢体。最近的研究建立了分子模型，整合了一组可能在控制再生细胞可塑性方面具有重要功能的基因。其中包括Msx1，它在体外可诱导哺乳动物肌管去分化。在此，我们使用酵母双杂交系统筛选了蝾螈Msx1的相互作用伙伴。构建了一个来自5日龄伤口表皮和下层组织的双杂交cDNA文库，其中包含超过2×10⁶个cDNA，并用于筛选。分离并测序了34个所得的cDNA克隆。然后，我们将分离克隆的序列与蝾螈EST数据库（BLASTn）中注释的EST重叠群进行比较，以鉴定推定的直系同源物。随后，我们使用BLASTx在非冗余的NCBI序列数据库中搜索所有未匹配的克隆序列。这是首次将酵母双杂交系统应用于蝾螈动物模型，并成功用于筛选在两栖动物肢体再生背景下与Msx1相互作用的蛋白质。
