Corsini Michela, Moroni Emanuela, Ravelli Cosetta, Grillo Elisabetta, Presta Marco, Mitola Stefania
Department of Molecular and Translational Medicine, University of Brescia, 25123 Brescia, Italy.
Methods Protoc. 2020 Nov 21;3(4):80. doi: 10.3390/mps3040080.
The chick embryo chorioallantoic membrane (CAM) represents a powerful in vivo model to study several physiological and pathological processes including inflammation and tumor progression. Nevertheless, the possibility of deepening the molecular processes in the CAM system is biased by the absence/scarcity of chemical and biological reagents, designed explicitly for avian species. This is particularly true for transcriptional factors, proteinaceous molecules that regulate various cellular responses, including proliferation, survival, and differentiation. Here, we propose a detailed antibody-independent protocol to visualize the activation and nuclear translocation of transcriptional factors in cells or in tissues of different animal species. As a proof of concept, DNA/cAMP response element-binding protein (CREB) interaction was characterized on the CAM tissue using oligonucleotides containing the palindromic binding sequence of CREB. Scrambled oligonucleotides were used as controls. In situ DNA/protein interaction protocol is a versatile method that is useful for the study of transcription factors in the cell and tissue of different origins.
鸡胚绒毛尿囊膜（CAM）是一种强大的体内模型，可用于研究多种生理和病理过程，包括炎症和肿瘤进展。然而，由于缺乏专门为鸟类设计的化学和生物试剂，深入研究CAM系统中分子过程的可能性受到了限制。转录因子尤其如此，转录因子是调节各种细胞反应（包括增殖、存活和分化）的蛋白质分子。在这里，我们提出了一种详细的非抗体依赖方案，用于可视化不同动物物种细胞或组织中转录因子的激活和核转位。作为概念验证，使用含有CREB回文结合序列的寡核苷酸在CAM组织上表征了DNA/环磷酸腺苷反应元件结合蛋白（CREB）的相互作用。随机寡核苷酸用作对照。原位DNA/蛋白质相互作用方案是一种通用方法，可用于研究不同来源细胞和组织中的转录因子。
