Strategies to mitigate transgene–promoter interactions

Abstract

SummaryThe 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.

Analysis of the enhancer-blocking function of the TBS element from Petunia hybrida in transgenic Arabidopsis thaliana and Nicotiana tabacum

“…Another means of averting enhancer-promoter communication in transgenic constructs is the use of enhancer-blocking insulators, which impede such interactions when situated between enhancer and promoter, and are commonly used during mammalian cell transfection experiments (Steinwaerder and Lieber 2000;Ye et al 2003). Unfortunately, relatively little is known concerning these elements in plants; however, several sequences exhibiting enhancer-blocking activity in plants have been identified in recent years (Gudynaite-Savitch et al 2009;Singer et al 2010b;van der Geest and Hall 1997). One such sequence is the TBS fragment from P. hybrida, which has been shown to impede inappropriate enhancer-promoter interference in transgenic Arabidopsis when situated in the reverse orientation (relative to the sequence deposited in GenBank) between the 35S enhancer and the flower-specific AGIP target promoter .…”

“…Several methods have been proposed to prevent such interactions, including the inclusion of a spacer DNA fragment between enhancer and promoter, or the use of promoters that contain only weak enhancers. However, these techniques have been shown to be relatively unpredictable and their effectiveness often varies from construct to construct (Gudynaite-Savitch et al 2009). Another means of averting enhancer-promoter communication in transgenic constructs is the use of enhancer-blocking insulators, which impede such interactions when situated between enhancer and promoter, and are commonly used during mammalian cell transfection experiments (Steinwaerder and Lieber 2000;Ye et al 2003).…”

“…This phenomenon is particularly prevalent in transgenic plants harboring constructs in which the enhancer from the CaMV 35S promoter (Odell et al 1988) is in relatively close proximity to the promoter of another transgene, and results in both a loss of specificity and an increase in the level of expression induced by the transgenic promoter Zheng et al 2007). Interestingly, this enhancer-mediated activation of proximal promoters appears to be a fairly common property of a wide range of enhancers, and as a result, has incited interest in the development of strategies with which to prevent such interactions (Gudynaite-Savitch et al 2009;Singer et al 2010b).…”

“…Since not all enhancers possess the same activation potential (Gudynaite-Savitch et al 2009), a possible solution to this problem is to generate transgenic constructs utilizing enhancers that do not have an effect on the tissue-specificity or strength of nearby promoters. However, promoters vary in their responsiveness to the activation potential of each enhancer, suggesting that one single enhancer would not necessarily solve the problem.…”

Transformation of <i>Lesquerella Fendleri</i> with the New Binary Vector pGPro4-35S

“…The pGPro4 vector is well-suited for promoter characterization in dicot plants because it avoids promiscuous promoter interactions and/or interference that have been shown to occur between the CaMV35S promoter/enhancer used to control selectable marker expression and nearby promoters in other commonly used binary vectors (Gudynaite-Savitch et al, 2009;Hily et al, 2009;Singer et al, 2010;So et al, 2005;Zheng et al, 2007). The nopaline synthase (nos) promoter has been demonstrated to not alter the expression of nearby organ or tissue-specific promoters (Gudynaite-Savitch et al, 2009;So et al, 2005;Zheng et al, 2007) and thus the nos promoter was utilized to control selectable marker expression in pGPro4 ( Figure 1A). The T-DNA also contains loxP recombinase recognition sites flanking the nos promoter and hygromycin phosphotransferase II (hptII) gene, thus enabling Cre recombinase-mediated site-specific excision of the selectable marker gene, to produce marker-free transgenic plants.…”