Arabidopsis transformation with large bacterial artificial chromosomes

The study of a gene's function requires, in many cases, the ability to reintroduce the gene of interest or its modified version back into the organism of choice. One potential caveat of this approach is that not only the coding region but also the regulatory sequences of a gene should be included in the corresponding transgenic construct. Even in species with well-annotated genomes, such as Arabidopsis, it is nearly impossible to predict which sequences are responsible for the proper expression of a gene. One way to circumvent this problem is to utilize a large fragment of genomic DNA that contains the coding region of the gene of interest and at least 5-10 kb of flanking genomic sequences. To facilitate these types of experiments, libraries harboring large genomic DNA fragments in binary vectors have been constructed for Arabidopsis and several other plant species. Working with these large clones, however, requires some special precautions. In this chapter, we describe the experimental procedures and extra cautionary measures involved in the identification of the clone containing the gene of interest, its transfer from E. coli to Agrobacterium, and the generation, verification, and analysis of the corresponding transgenic plants.