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Use of large-scale expression cloning screens in the Xenopus laevis tadpole to identify gene function
T C Grammer1, K J Liu, F V Mariani
1Department of Molecular and Cell Biology, University of California at Berkeley, 401 Barker Hall, Berkeley, California 94720, USA.
Abstract:
We have conducted an expression cloning screen of approximately 50, 000 cDNAs from a tadpole stage Xenopus laevis cDNA library to functionally identify genes affecting a wide range of cellular and developmental processes. Fifty-seven cDNAs were isolated for their ability to alter gross tadpole morphology or the expression patterns of tissue-specific markers. Thirty-seven of the cDNAs have not been previously described for Xenopus, and 15 of these show little or no similarity to sequences in the NCBI database. The screen and the identified genes are presented in this paper to demonstrate the power, ease, speed, and flexibility of expression cloning in the X. laevis embryo. Future screens such as this one can be done on a larger scale and will complement the sequence-based screens and genome-sequencing projects which are producing a large body of novel genes without ascribed functions.
Insights
Researchers screened Xenopus laevis cDNAs to identify genes impacting development. This expression cloning approach rapidly uncovered novel genes influencing tadpole morphology and tissue development.
Area of Science:
- Developmental Biology
- Molecular Biology
- Genetics
Background:
- Functional genomics relies on identifying gene functions.
- Expression cloning is a powerful method for discovering genes with specific biological activities.
- The Xenopus laevis embryo provides a versatile model for studying early development.
Purpose of the Study:
- To perform an expression cloning screen of Xenopus laevis cDNAs.
- To identify novel genes that regulate cellular and developmental processes.
- To demonstrate the utility of expression cloning in Xenopus.
Main Methods:
- A large-scale expression cloning screen was performed using approximately 50,000 Xenopus laevis cDNAs.
- Functional screening was based on observing alterations in tadpole morphology and tissue-specific marker expression.
- Isolated cDNAs were analyzed for novelty and sequence similarity.
Main Results:
- Fifty-seven cDNAs were isolated for their ability to modify tadpole development.
- Thirty-seven of the isolated cDNAs were novel to Xenopus.
- Fifteen novel cDNAs showed minimal similarity to existing sequences in the NCBI database.
Conclusions:
- Expression cloning in Xenopus laevis embryos is an effective, rapid, and flexible method for gene discovery.
- This approach successfully identified numerous genes involved in developmental processes.
- Expression cloning complements large-scale sequencing projects by assigning functions to novel genes.