Abstract
Genomic DNA libraries are a valuable source of large constructs that can contain all the regulatory elements necessary for recapitulating wild-type gene expression when introduced into animal genomes as a transgene. Such clones can be directly used in complementation studies. In combination with recombineering manipulation, the tagged genomic clones can serve as faithful in vivo gene activity reporters that enable studies of tissue specificity of gene expression, subcellular protein localization, and affinity purification of complexes. We present a detailed protocol for generating an unbiased genomic library in a custom pFlyFos vector that is optimized for liquid culture recombineering manipulation and site-specific transgenesis of fosmid-size loci across different Drosophila species. The cross-species properties of the library can be used, for example, to establish the specificity of RNAi phenotypes or to selectively introgress specific genomic loci among different Drosophila species making it an ideal tool for experimental evolutionary studies. The FlyFos system can be easily adapted to other organisms.
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Ejsmont, R.K., Bogdanzaliewa, M., Lipinski, K.A., Tomancak, P. (2012). Production of Fosmid Genomic Libraries Optimized for Liquid Culture Recombineering and Cross-Species Transgenesis. In: Orgogozo, V., Rockman, M. (eds) Molecular Methods for Evolutionary Genetics. Methods in Molecular Biology, vol 772. Humana Press. https://doi.org/10.1007/978-1-61779-228-1_25
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DOI: https://doi.org/10.1007/978-1-61779-228-1_25
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Publisher Name: Humana Press
Print ISBN: 978-1-61779-227-4
Online ISBN: 978-1-61779-228-1
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