The main task of vertebrate post-genome era is to decipher the function of genes, while gene trapping and insertional mutagenesis are key approaches for this purpose. Zebrafish (Danio rerio) has become an ideal animal model for studies of development and genetics due to its advantageous characteristics such as easy feeding, short breeding cycle, high fecundity and external embryonic development, but embryonic stem cells and embryonic stem cell-based gene knockout technology in zebrafish remain to be developed. Transposon-mediated gene trapping and insertional mutagenesis can provide a new way for large-scale screening of zebrafish mutants and elucidation of trapped gene functions.
A research group led by Prof. Cui Zongbin from Institute of Hydrobiology, Chinese Academy of Sciences(IHB) has designed and constructed novel transposon-based gene- and poly(A)-trap vectors on the basis of in-depth understanding about Sleeping Beauty (SB) transposition mechanism and in combination with latest results of gene trappingresearches. These two vectors were utilized for the capture of functional genes in cultured cells and developing embryos of zebrafish.
Main results of these studies include: 1) Activities of all elements in trapping vectors were tested in HeLa cells and zebrafish embryos. 2) A large number of genes in human HeLa cells were captured and the transcriptional expression of these genes was efficiently disrupted by elements in gene- and poly(A)-trap vectors. 3) The tilapia heat shock protein 70 promoter can drive the inducible expression of SB gene, which allows the remobilization of integrated trapping cassettes and thus improves the efficiency of finding genes. 4) The inducible TiHsp70-SB11 cassette can be excised by the Cre/LoxP system for the stabilization of mutants; 5) The use of trap vectors has successfully captured a number of zebrafish genes, significantly interrupted their transcriptional expression, and obtained a number of zebrafish mutant lines.
These findings has provided a new technology platform for investigation of gene functions in zebrafish and laid a solid foundation for the establishment of a zebrafish mutant library.The above researches were supported by the National Basic Research Program of China and the National Natural Science Foundation of China, and mainly performed by a PhD student Song Guili.
The results have been published in PLoS ONE (http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0044123) and Journal of Genetics & Genomics (http://dx.doi.org/10.1016/j.jgg.2012.05.010).
