In vivo modeling of the morbid human genome using Danio rerio.
In vivo modeling of the morbid human genome using Danio rerio.
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DOI：
10.3791/50338
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发表时间：
2013-08-24
期刊：
影响因子：
--
通讯作者：
Katsanis N
中科院分区：
文献类型：
--
作者：
Niederriter AR;Davis EE;Golzio C;Oh EC;Tsai IC;Katsanis N
在这里，我们提出的方法，用于开发检测查询潜在的临床显着的非同义变化，在斑马鱼体内互补。斑马鱼（Danio rerio）是一种有用的动物系统，因为它们的实验易处理性;胚胎是透明的，便于观察，经历快速的体外发育，并且可以进行遗传操作。这些方面已经允许在胚胎发生，分子过程和形态发生信号的分析中取得重大进展。总之，这种脊椎动物模型的优点使斑马鱼非常适合模拟儿科疾病的发育缺陷，在某些情况下，成人发病的疾病。由于斑马鱼基因组与人类基因组高度保守（约70%同源），因此有可能在斑马鱼中重现人类疾病状态。这是通过注射突变体人mRNA以诱导显性失活或获得功能等位基因，或利用吗啉代（MO）反义寡核苷酸抑制基因以模拟功能丧失变体来实现的。通过与加帽的人mRNA互补MO诱导的表型，我们的方法能够基于突变mRNA拯救可测量的生理相关表型的能力来解释突变对人蛋白质序列的有害影响。人类疾病等位基因的建模通过在1-4细胞阶段用MO和/或人mRNA显微注射斑马鱼胚胎，并在受精后（dpf）长达7天进行表型分析来进行。这种一般策略可以扩展到广泛的疾病表型，如以下方案所示。我们提出了我们建立的形态发生信号，颅面，心脏，血管完整性，肾功能和骨骼肌疾病表型，以及其他模型。
Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated. These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.
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影响因子：
14.8
作者：
Kumar, Prateek;Henikoff, Steven;Ng, Pauline C.
通讯作者：
Ng, Pauline C.
影响因子：
4
作者：
Marion, Vincent;Stutzmann, Fanny;Dollfus, Helene
通讯作者：
Dollfus, Helene
影响因子：
30.8
作者：
Gerdes, Jantje M.;Liu, Yangfan;Katsanis, Nicholas
通讯作者：
Katsanis, Nicholas
影响因子：
4.4
作者：
Bohnsack, Brenda L.;Kasprick, Daniel S.;Kahana, Alon
通讯作者：
Kahana, Alon
影响因子：
2.5
作者：
KIMMEL, CB;BALLARD, WW;SCHILLING, TF
通讯作者：
SCHILLING, TF