Xavier Pedro L P, Senhorini José A, Pereira-Santos Matheus, Fujimoto Takafumi, Shimoda Eduardo, Silva Luciano A, Dos Santos Silvio A, Yasui George S
National Center for Research and Conservation of Continental Fish, Chico Mendes Institute of Biodiversity ConservationPirassununga, Brazil.
Aquaculture Center, Sao Paulo State UniversityJaboticabal, Brazil.
Front Genet. 2017 Sep 25;8:131. doi: 10.3389/fgene.2017.00131. eCollection 2017.
The production of triploid yellowtail tetra is a key factor to obtain permanently sterile individuals by chromosome set manipulation. Flow cytometric analysis is the main tool for confirmation of the resultant triploids individuals, but very few protocols are specific for species. The current study has developed a protocol to estimate DNA content in this species. Furthermore, a protocol for long-term storage of dorsal fins used for flow cytometry analysis was established. The combination of five solutions with three detergents (Nonidet P-40 Substitute, Tween 20, and Triton X-100) at 0.1, 0.2, and 0.4% concentration was evaluated. Using the best solution from this first experiment, the addition of trypsin (0.125, 0.25, and 0.5%) and sucrose (74 mM) and the effects of increased concentrations of the detergents at 0.6 and 1.2% concentration were also evaluated. After adjustment of the protocol for flow cytometry, preservation of somatic tissue or isolated nuclei was also evaluated by freezing (at -20°C) and fixation in saturated NaCl solution, acetic methanol (1:3), ethanol, and formalin at 10% for 30 or 60 days of storage at 25°C. Flow cytometry analysis in yellowtail tetra species was optimized using the following conditions: lysis solution: 9.53 mM MgCl.7H0; 47.67 mM KCl; 15 mM Tris; 74 mM sucrose, 0.6% Triton X-100, pH 8.0; staining solution: Dulbecco's PBS with DAPI 1 μg mL; preservation procedure: somatic cells (dorsal fin samples) frozen at -20°C. Using this protocol, samples may be stored up to 60 days with good accuracy for flow cytometry analysis.
三倍体双线察的产生是通过染色体组操作获得永久不育个体的关键因素。流式细胞术分析是确认所得三倍体个体的主要工具，但针对该物种的特定方案非常少。当前研究已开发出一种用于估算该物种DNA含量的方案。此外，还建立了用于流式细胞术分析的背鳍长期保存方案。对五种溶液与三种去污剂（去氧胆酸钠、吐温20和曲拉通X-100）以0.1%、0.2%和0.4%浓度的组合进行了评估。使用首次实验中最佳的溶液，还评估了添加胰蛋白酶（0.125%、0.25%和0.5%）和蔗糖（74 mM）以及将去污剂浓度提高到0.6%和1.2%的效果。在调整流式细胞术方案后，还通过在-20°C冷冻以及在饱和NaCl溶液、冰醋酸甲醇（1:3）、乙醇和10%福尔马林中固定来评估体细胞组织或分离细胞核在25°C下保存30或60天的情况。使用以下条件优化了双线察的流式细胞术分析：裂解液：9.53 mM MgCl₂·7H₂O；47.67 mM KCl；15 mM Tris；74 mM蔗糖，0.6%曲拉通X-100，pH 8.0；染色液：含1 μg/mL DAPI的杜氏磷酸盐缓冲液；保存程序：体细胞（背鳍样本）在-20°C冷冻。使用该方案，样本可保存长达60天，用于流式细胞术分析时具有良好的准确性。
