Direct Chromosome Preparation Method in Avian Embryos for Cytogenetic Studies: Quick, Easy and Cheap

Abstract

Avian cell culture is widely applied for cytogenetic studies, the improvement of which increasingly allows for the production of high-quality chromosomes, essential to perform both classical and molecular cytogenetic studies. Among these approaches, there are two main types: fibroblast and bone marrow culture. Despite its high cost and complexity, fibroblast culture is considered the superior approach due to the quality of the metaphases produced. Short-term bone marrow cultivation provides more condensed chromosomes but nonetheless is quicker and easier. In the search for a quicker, cheaper way to prepare metaphases without losing quality, the present work developed a novel, widely applicable protocol for avian chromosome preparation. Twenty-one bird embryos from distinct families were sampled: Icteridae, Columbidae, Furnariidae, Estrildidae, Thraupidae, Troglodytidae and Ardeidae. The protocol was based on a combination of modified fibroblast culture and bone marrow cultivation, taking the advantages of both. The results show that all species consistently presented good mitotic indexes and high-quality chromosomes. Overall, the application of this protocol for bird cytogenetics can optimize the time, considering that most fibroblast cultures take at least 3 days and often much longer. However, our protocol can be performed in 3 h with a much-reduced cost of reagents and equipment.

Microchromosome BAC-FISH Reveals Different Patterns of Genome Organization in Three Charadriiformes Species

“…Metaphase chromosomes were obtained according to standard procedures involving exposure to colcemid (1 h, 37 °C), hypotonic treatment (0.075 M KCl, 15 min, 37 °C), and fixation with methanol/acetic acid (3:1). V. chilensis species was also sampled by the direct chromosome preparation method, where embryo cells were dissociated by 2 mL of trypsin 0.25% EDTA for approximately 10 min, then the sample was placed in 10 mL of RPMI 1040 medium pre warmed at 37 °C with three drops of colchicine 0.05% and incubated for 1 h at 37 °C, followed by hypotonic treatment and fixation [ 25 ].…”