In Situ DNA/Protein Interaction Assay to Visualize Transcriptional Factor Activation

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.

Pentraxin 3 Inhibits the Angiogenic Potential of Multiple Myeloma Cells

“…Alginate beads (5 μL) containing vehicle or KMS-11 PTX3 cells (40 × 10 4 cells/implant) with or without DOXA (200 μg/mL) were placed onto the top of chicken embryo CAMs at day 11 of incubation. After 72 h, newly formed blood vessels were quantified as described [ 22 , 23 , 24 ].…”

Simultaneously characterization of tumoral angiogenesis and vasculogenesis in stem cell-derived teratomas

“…At day 4 post incubation (p.i.) the shells were covered with a transparent adhesive tape and a small window sawed with scissors [30]. At day 7 p.i., drilled plastic coverslips were positioned on the surface of the CAM in a well vascularized area.…”