Expression cloning screening of a unique and full-length set of cDNA clones is an efficient method for identifying genes involved in Xenopus neurogenesis
Expression cloning screening of a unique and full-length set of cDNA clones is an efficient method for identifying genes involved in Xenopus neurogenesis
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对一组独特的全长 cDNA 克隆进行表达克隆筛选是鉴定非洲爪蟾神经发生相关基因的有效方法
DOI：
10.1016/j.mod.2004.11.002
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发表时间：
2005
影响因子：
2.6
通讯作者：
N. Papalopulu
中科院分区：
文献类型：
--
作者：
J. Voigt;Jun;Mike Gilchrist;E. Amaya;N. Papalopulu
功能筛选，其中大量的cDNA克隆的某些生物活性测定，是一个有用的工具，在阐明基因功能。在非洲爪蟾中，功能筛选的获得是通过池筛选进行的，即从数千到100个cDNA克隆组中体外转录的RNA被注射到早期胚胎中。一旦在池中检测到某个活动，就通过同胞选择来识别活动克隆。这种筛选本质上偏向于强效基因，其RNA活性较低。为了提高功能筛选的灵敏度和效率，我们对100,000个原肠和神经细胞cDNA克隆进行了生物信息学处理和EST测序，以创建一个独特的全长约2500个克隆。减少冗余并从开始克隆集中排除截断的克隆减少了要筛选的克隆的总数，从而使我们能够将池大小减少到每个池只有8个克隆。我们报告说，筛选这个克隆集的效率比从相同文库衍生的冗余集高5倍。我们从这组样本中筛选了960个cDNA克隆，寻找与神经发生有关的基因。我们描述了18个单克隆的过表达表型，其中大多数显示出以前未表征的表型，其中一些是完全新颖的。原位杂交分析表明，大量这些基因在神经组织中特异性表达。这些结果证明了一套独特的全长cDNA克隆在揭示发育途径中的参与者方面的有效性。
Functional screens, where a large numbers of cDNA clones are assayed for certain biological activity, are a useful tool in elucidating gene function. In Xenopus, gain of function screens are performed by pool screening, whereby RNA transcribed in vitro from groups of cDNA clones, ranging from thousands to a hundred, are injected into early embryos. Once an activity is detected in a pool, the active clone is identified by sib-selection. Such screens are intrinsically biased towards potent genes, whose RNA is active at low quantities. To improve the sensitivity and efficiency of a gain of function screen we have bioinformatically processed an arrayed and EST sequenced set of 100,000 gastrula and neurula cDNA clones, to create a unique and full-length set of approximately 2500 clones. Reducing the redundancy and excluding truncated clones from the starting clone set reduced the total number of clones to be screened, in turn allowing us to reduce the pool size to just eight clones per pool. We report that the efficiency of screening this clone set is five-fold higher compared to a redundant set derived from the same libraries. We have screened 960 cDNA clones from this set, for genes that are involved in neurogenesis. We describe the overexpression phenotypes of 18 single clones, the majority of which show a previously uncharacterised phenotype and some of which are completely novel. In situ hybridisation analysis shows that a large number of these genes are specifically expressed in neural tissue. These results demonstrate the effectiveness of a unique full-length set of cDNA clones for uncovering players in a developmental pathway.
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影响因子：
10.5
作者：
Baker, JC;Harland, RM
通讯作者：
Harland, RM
影响因子：
2.7
作者：
McKay, RM;Peters, JM;Graff, JM
通讯作者：
Graff, JM
非洲爪蟾同源盒基因孪生介导 Goosecoid 的 Wnt 诱导，从而建立了 Spemann 组织体。
DOI：
10.1242/dev.124.23.4905
发表时间：
1997
期刊：
Development (Cambridge, England)
影响因子：
--
作者：
Laurent,MN;Blitz,IL;Hashimoto,C;Rothbächer,U;Cho,KW
通讯作者：
Cho,KW
影响因子：
20.3
作者：
Chen, E;Hermanson, S;Ekker, SC
通讯作者：
Ekker, SC
影响斑马鱼胚胎发生的突变的遗传筛查。
DOI：
10.1242/dev.123.1.37
发表时间：
1996
期刊：
Development (Cambridge, England)
影响因子：
--
作者：
Driever,W;Solnica-Krezel,L;Schier,AF;Neuhauss,SC;Malicki,J;Stemple,DL;Stainier,DY;Zwartkruis,F;Abdelilah,S;Rangini,Z;Belak,J;Boggs,C
通讯作者：
Boggs,C