Plant Biotechnology Journal (2009) 7, pp. 472– 485 doi: 10.1111/j.1467-7652.2009.00416.x © 2009 Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada 472 Journal compilation © 2009 Blackwell Publishing Ltd Blackwell Publishing Ltd Oxford, UK PBI Plant Biotechnology Journal 1467-7644 1467-7652 © 2009 Blackwell Publishing Ltd XXX Original Article Mitigation of transgene–promoter interactions Loreta Gudynaite-Savitchet al. Strategies to mitigate transgene–promoter interactions Loreta Gudynaite-Savitch 1, †, Douglas A. Johnson 1, * and Brian L. A. Miki 2 1 Ottawa-Carleton Institute of Biology, University of Ottawa, PO Box 450, Station A, Ottawa, ON, Canada, K1N 6N5 2 BioProducts & BioProcesses Research Branch, Agriculture and Agri-Food Canada, Ottawa, ON, Canada, K1A OC6 Summary The expression pattern of tissue-specific promoters in transgenes can be influenced by promoter/enhancer elements employed for the expression of selectable marker genes or elements found in DNA flanking the insertion site. We have developed an analytical system in Arabidopsis thaliana to investigate strategies useful in blocking or reducing nonspecific interactions. These experiments confirm that the DNA configuration and the insertion of spacer DNA aid in the appropriate expression of tissue-specific promoters. It is also demonstrated that the novel tobacco cryptic promoter (tCUP), when used to replace the cauliflower mosaic virus (CaMV) 35S promoter/enhancer, does not show nonspecific interactions. Furthermore, it is shown that insulators isolated from yeast and animals may have potential application in plants. Our results may allow the design of strategies that, individually or in combination, can be used to minimize nonspecific interactions and to design vectors for individual tissue-specific promoters. Received 3 November 2008; revised 20 January 2009; accepted 21 January 2009. *Correspondence (fax 1-613-562-5486; e-mail: johnson@uottawa.ca) †Present address: Iogen Corporation, 310 Hunt Club Rd., Ottawa, ON, Canada, K1V 1C1 Keywords: cauliflower mosaic virus (CaMV) 35S promoter, promoter– enhancer interaction, tCUP promoter, tissue-specific expression, tissue- specific promoter, transgenic plants. Introduction Transgenic plants with targeted transgene expression play a central role in the study of plant development and plant biotechnology. Initially, expression was achieved throughout the plant using constitutive promoters, such as the 35S promoter from cauliflower mosaic virus (CaMV). Although this strategy remains valid for some applications, recent effort has focused on the use of tissue-specific promoters that restrict the transgene expression to targeted tissues only. This approach has been employed for the development of male-sterile transgenic plants using tapetum-specific promoters for the expression of the barnase gene (Denis et al., 1993; Jagannath et al., 2001), and for the generation of tomato plants with delayed ripening or modified carotenoid contents using E-8-fruit-ripening-specific polygalacturonase and fruit-specific phytoene desaturase promoters up-regulated during fruit ripening (Rosati et al., 2000; Krasnyanski et al., 2001; Fraser et al., 2002). The modification of seed storage compounds has used a variety of endosperm- and seed- specific promoters, such as the rice glutelin-1 (Goto et al., 1999; Datta et al., 2003; Paine et al., 2005), soybean lectin (Guerche et al., 1990) and Brassica cruciferin or napin (Lee et al., 1991; Knutzon et al., 1992; Shewmaker et al., 1999; Ponstein et al., 2002) promoters. The production of pharma- ceutical proteins in seeds, where they can remain stable and functional for several years, is a very attractive application. Endosperm- and seed-specific promoters are used in the production of biopharmaceuticals, edible vaccines and antibodies in several cereal and legume species (Stoger et al., 2005). Although most examples result from the expression of a single transgene, future efforts may involve the introduction of multiple genes; for example, to redirect metabolic flow to produce new oils (Wu et al., 2005) requires the introduction of multiple genes on a single vector. The successful selection of transgenic plants requires strong constitutive promoters to express selectable marker genes to avoid unnecessary and time-consuming screening for the trait of interest. Over 50 different selectable marker gene systems have been described; however, those employing kanamycin (neomycin phosphotransferase II, nptII) or hygromycin (hygromycin phosphotransferase II , hptII ) resistance (reviewed by Miki and McHugh, 2004), expressed from the CaMV 35S promoter or the nopaline synthase (nos) promoter of Agrobacterium tumefaciens, predominate. The CaMV 35S promoter (Covey et al., 1981) is widely used to drive the