Abstract
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.
Highlights
The chick embryo chorioallantoic membrane (CAM) is an extraembryonic membrane that exhibits an extensive and well-organized capillary network composed of narrow arteries and veins and of lymphatic vessels that envelops the chick embryo [1,2,3,4]
Activated cAMP response element-binding protein (CREB) binds a highly conserved octameric palindromic DNA sequence (5 -TGACGTCA-3 ), named cAMP response elements (CRE), or a slight variant that are located within the promoter or enhancer regions of several viral and cellular genes [24]
All the antibody-based assays aimed to investigate the spatial-temporal activation of CREB require protein-specific as well as species-specific antibodies
Summary
The chick embryo chorioallantoic membrane (CAM) is an extraembryonic membrane that exhibits an extensive and well-organized capillary network composed of narrow arteries and veins and of lymphatic vessels that envelops the chick embryo [1,2,3,4]. The easy access for experimental manipulation and the low cost make this system a valid alternative to other in vivo models [16,17] Despite these advantages, there are some limitations in the use of CAM in experimentation, especially for antibody-based assays. We propose a detailed oligonucleotide-based protocol to study CREB activation directly in avian tissues. This in situ DNA/protein interaction assay is suitable for the visualization of the spatial–temporal activation of transcriptional factors other than CREB and can be applied to cells and tissues of various animal species in different experimental models
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