早期开发期间海葵基因分型
Summary
该协议的目标是在胃化过程中基因型海葵奈马托斯特拉静脉曲张,而不牺牲胚胎。
Abstract
这里描述的是基于PCR的协议基因型的胃肠阶段胚胎的蚂蚁阴极内人内马托斯特拉vesisis,而不牺牲动物的生命。在体外受精和脱胶后,酶在室温下发育24小时,达到早至中气期。然后,将胃粒胚胎放在含有海水的培养皿中的甘蔗胶床上。在解剖显微镜下,钨针用于从每个胚胎中手术分离一个腹腔组织片段。手术后胚胎可以愈合并继续发育。基因组DNA从分离的组织片段中提取,并用作位点特异性PCR的模板。基因型可以根据PCR产品的大小或存在/不存在的等位基因特异性PCR产品来确定。手术后胚胎然后根据基因型进行分类。整个基因分型过程的持续时间取决于要筛选的胚胎数量,但它至少需要4~5小时。该方法可用于识别来自基因异质胚胎种群的敲除突变体,并在发育过程中分析表型。
Introduction
仙人掌代表一组不同的动物,包括水母、珊瑚和海葵。它们是二叶虫,由细胞外基质(中细胞体)分离的内皮和内皮组成。Cnidaria是一个姐妹团体,以种像比拉提利亚,传统的动物模型,如果蝇和穆斯属于1。此外,被认为发生在2月2日寒武纪前,Cnidaria-Bilateria发散。因此,对仙人掌和双栖教徒的比较研究对于深入了解他们最近共同祖先的生物学至关重要。最近,比较基因组学显示,仙人掌和双子食者共享许多发育工具包基因,如notch和bHLH,这意味着他们的共同祖先已经拥有这些基因3。然而,这些发育工具包基因在Cnidaria和Bilateria的最后共同祖先中的作用相对而言,还不太了解。为了解决这个问题,研究这些深保守的基因在仙人掌中如何发挥作用是至关重要的。
新兴的仙人掌遗传模型之一是蚂蚁内马托斯特拉的维森斯。其基因组已测序3,各种基因工具,包括形态介导基因敲除,梅甘细胞介导的转基因,CRISPR-Cas9介导基因敲和敲,现在可用于这种动物。此外,内马托斯特拉的发展也相对来说也比较了解。在胚胎生成过程中,胃化发生于阴道4,胚胎发育成自由游泳的平面幼虫。之后,平面图转化为带嘴和圆角触角的环状息肉。息肉然后增长,并达到性成熟。
CRISPR-Cas9介导的靶向诱变现因现在经常用于研究内马托斯特拉维克塞西5、6、7、8、9的基因功能。为了在Nematostella产生敲除突变体,一种含有原点特异性单导RNA和内分酶Cas9蛋白的鸡尾酒首先被注射到未受精或受精的卵子中,以产生通常显示的F0创始人动物马赛克主义。F0动物随后被提升到性成熟,并相互交叉,以产生F1种群,其中一个子集可能是敲除突变体6。或者,性成熟的F0动物可以与野生型动物交叉,以产生F1杂物动物,而F1杂物携带一个敲击等位基因在兴趣点,然后可以交叉彼此产生F2后代,四分之一其中预计将被敲除突变体5。这两种方法都需要一种方法来识别来自基因异质种群的敲除突变体。息触角可用于提取基因组DNA进行基因分型6,7。然而,在研究相关基因的发育功能和突变胚胎未达到息肉阶段的情况下(即,由于与突变相关的幼虫致死性),需要在早期发现敲除突变体。这里描述的是基于PCR的方案基因型个体动物在气态阶段不牺牲动物,这使得识别从基因异质的胚胎种群敲除突变体。整个基因分型过程的持续时间取决于要筛选的胚胎数量,但它至少需要4-5小时。
Protocol
Representative Results
Discussion
这里描述了一种基于PCR的协议,用于基因型单个海合明胚胎,而不牺牲动物。产卵和脱硫后,受精卵可以发育成胃卵。每个胃肠道胚胎的腹腔区域被手术切除,分离的腹腔组织用于后续基因组DNA提取,而剩余的手术后胚胎愈合并继续发育。然后,gDNA提取物用于PCR测定,以确定每个胚胎的基因型。该方法利用海葵胚胎的口腔半部分调节和发育10,11和大多数胚胎的能力(>90%)<sup class="xref…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢匿名评论者对手稿的早期版本的评论,该版本改进了手稿。这项工作得到了阿肯色大学的资金支持。
Materials
| Drosophila Peltier Refrigerated Incubator | Shellab | SRI6PF | Used for spawning induction |
| Instant ocean sea salt | Instant ocean | 138510 | |
| Brine shrimp cysts | Aquatic Eco-Systems, Inc. | BS90 | |
| L-Cysteine Hydrochloride | Sigma Aldrich | C7352 | |
| Standard Orbital Shaker, Model 3500 | VWR | 89032-092 | |
| TRIS-HCl, 1M, pH8.0 | QUALITY BIOLOGICAL | 351-007-01 | |
| Potassium chloride | VWR | BDH9258 | |
| EDTA, 0.5M pH8 | VWR | BDH7830-1 | |
| Tween 20 | Sigma Aldrich | P9416 | |
| Nonidet-P40 Substitute | US Biological | N3500 | |
| Proteinase K solution (20 mg/mL), RNA grade | ThermoFisher | 25530049 | |
| Agarose | VWR | 710 | |
| Micro Dissecting needle holder | Roboz | RS-6060 | |
| Tungsten dissecting needle | Roboz | RS-6063 | |
| PCR Eppendorf Mastercycler Thermal Cyclers | Eppendorf | E6336000024 | |
| Phusion High-Fidelity DNA polymerase | New England BioLabs | M0530L | |
| dNTP mix | New England BioLabs | N0447L | |
| GLWamide universal forward primer | 5’- CATGCGGAGACCAAGCGCAAGGC-3’ | ||
| Reverse primer specific to glw-a | 5’-CCAGATGCCTGGTGATAC-3’ | ||
| Reverse primer specific to glw-c | 5’- CGGCCGGCGCATATATAG-3’ |
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