Production of haploid zebrafish embryos by in vitro fertilization.
Production of haploid zebrafish embryos by in vitro fertilization.
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DOI：
10.3791/51708
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发表时间：
2014-07-14
期刊：
影响因子：
--
通讯作者：
Wingert RA
中科院分区：
文献类型：
--
作者：
Kroeger PT Jr;Poureetezadi SJ;McKee R;Jou J;Miceli R;Wingert RA
斑马鱼已经成为一种主流的脊椎动物模型，与许多学科的科学研究有关。斑马鱼特别适合于发育过程的正向遗传分析，因为它们的外部受精，胚胎大小，快速个体发育和光学清晰度-一系列特征，使直接观察从原肠胚形成到器官发生的事件具有基本的立体显微镜。此外，斑马鱼胚胎可以在单倍体状态下存活数天。在体外产生单倍体胚胎是突变分析的有力工具，因为它能够在亲本（P）代中诱变后鉴定第一代（F1）雌性携带者中存在的隐性突变等位基因。这种方法消除了提高多代（F2，F3等）的必要性。其涉及突变家族的育种，从而沿着节省研究者的时间，同时减少对斑马鱼群体空间、劳动力和饲养成本的需求。尽管斑马鱼在过去几十年中一直被用于进行正向筛选，但转基因和基因组编辑工具一直在稳步扩展。这些工具现在提供了大量的方法来为下一代筛选创建细致入微的检测，这些检测可用于进一步剖析驱动脊椎动物个体发育的基因调控网络。在这里，我们描述了如何制备单倍体斑马鱼胚胎。该方案可用于新的未来单倍体筛选，例如增强子和抑制子筛选，以解决在早期胚胎阶段形成的大量过程和组织的发育机制。
The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity – a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.
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通过全基因组测序快速纯合性作图和鉴定斑马鱼 ENU 诱导的突变。
影响因子：
3.7
作者：
Voz ML;Coppieters W;Manfroid I;Baudhuin A;Von Berg V;Charlier C;Meyer D;Driever W;Martial JA;Peers B
通讯作者：
Peers B
影响因子：
3.3
作者：
Bowen, Margot E.;Henke, Katrin;Harris, Matthew P.
通讯作者：
Harris, Matthew P.
DOI：
10.3791/1396
发表时间：
2009-06-30
期刊：
Journal of visualized experiments : JoVE
影响因子：
--
作者：
Walker, Charline;Walsh, Greg S;Moens, Cecilia
通讯作者：
Moens, Cecilia
影响因子：
2.5
作者：
Wingert, Rebecca A.;Davidson, Alan J.
通讯作者：
Davidson, Alan J.
影响因子：
2.5
作者：
KIMMEL, CB;BALLARD, WW;SCHILLING, TF
通讯作者：
SCHILLING, TF