Expression cloning screening of a unique and full-length set of cDNA clones is an efficient method for identifying genes involved in Xenopus neurogenesis

J. Voigt, J-A. Chen, M. Gilchrist, E. Amaya and N. Papalopulu

Mechanisms of Development 122:289-306 (2005) (Full Article, 1100 Kb).

ABSTRACT

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.

Back to The Amaya Lab Homepage