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.
TAC Transformation Arabidopsis T-DNA DNA deletions Electroporation Agrobacterium
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Alonso JM, Ecker JR (2006) Moving forward in reverse: genetic technologies to enable genome-wide phenomic screens in Arabidopsis. Nat Rev Genet 7:524–536PubMedCrossRefGoogle Scholar
Liu Y, Mitsukawa N, Vazquez-Tello A, Whittier RF (1995) Generation of a high-quality P1 library of Arabidopsis suitable for chromosome walking. Plant J 7:351–358CrossRefGoogle Scholar
Chang Y-C, Henriquez XH, Preuss DP, Copenhaver GC, Zhang HZ (2003) A plant-transformation-competent BIBAC library from the Arabidopsis thaliana Landsberg ecotype for functional and comparative genomics. Theor Appl Genet 106:269–276PubMedGoogle Scholar
Liu YG, Shirano Y, Fukaki H, Yanai Y, Tasaka M, Tabata S, Shibata D (1999) Complementation of plant mutants with large genomic DNA fragments by a transformation-competent artificial chromosome vector accelerates positional cloning. Proc Natl Acad Sci U S A 96:6535–6540PubMedCrossRefGoogle Scholar
Zhou R, Benavente LM, Stepanova AN, Alonso JM (2011) A recombineering-based gene tagging system for Arabidopsis. Plant J 66:712–723PubMedCrossRefGoogle Scholar
Farrand SK, O'Morchoe SP, McCutchan J (1989) Construction of an Agrobacterium tumefaciens C58 recA mutant. J Bacteriol 171:5314–5321PubMedGoogle Scholar
Sheng Y, Mancino V, Birren B (1995) Transformation of Escherichia coli with large DNA molecules by electroporation. Nucleic Acids Res 23:1990–1996PubMedCrossRefGoogle Scholar