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 ].…”

Highly Conserved Microchromosomal Organization in Passeriformes Birds Revealed via BAC-FISH Analysis

“…Metaphase chromosomes were obtained according to standard procedures involving colchicine exposure (1 h, 37 • C), hypotonic treatment (0.075 M KCl, 15 min, 37 • C), and methanol/acetic acid (3:1) fixation. A direct chromosome preparation method was also used for Double-collared Seedeater and Southern House Wren, in which embryonic cells were dissociated with 2 mL of trypsin 0.25% EDTA (Sigma-Aldrich, St. Louis, MO, USA) for approximately 10 min; samples were soon after placed in 10 mL of RPMI 1640 (GIBCO/Thermo Fisher Scientific, Burlington, MA, USA), pre-warmed to 37 • C, and then 3 drops of 0.05% colchicine were added, followed by incubation for 1 h at 37 • C before hypotonic treatment for 20 min and fixation with methanol/acetic acid (3:1) [25].…”

The Joint Meeting of the 23rd International Chromosome Conference (ICC) and the 24th International Colloquium in Animal Cytogenetics and Genomics (ICACG)

“…The studied species included representatives from the families Icteridae, Columbidae, Furnariidae, Estrildidae, Thraupidae, Troglodytidae and Ardeidae. Impressive results show consistently excellent mitotic indexes with high-quality chromosomes from all species in a time frame of only 3 h [6]. Finally, Mohammed Yusuf and colleagues present an exciting 3D ultrastructural imaging technique to visualise chromosomes using serial block-face scanning electron microscopy (SBFSEM).…”