Strategies for precise quantification of transgene expression levels over several generations in rice

Variation in transgene expression levels can result from uncontrolled differences in experimental protocols. Studies conducted over generations could, by their design, generate additional unwanted variation. To study sources of spurious variation, transgene expression levels were quantified over five homozygous generations in two independent transgenic rice lines created by particle bombardment. Both lines contained the same gus expression unit and had been shown to exhibit stable inheritance of transgene structure and expression. All plants were cultured and sampled using previously developed standardized protocols. Plants representative of each generation (T2, T3, T4, T5, T6) were grown either all together or across several different growth periods. GUS activity in plants from different generations was quantified either in the same assay or over multiple independent assays. Strategies in which plants were grown and phenotyped independently, significantly increased (up to 3-fold) extraneous variation in transgene expression level quantification, thus reducing the precision of molecular genetic studies and generating artefactual results in transgenic studies conducted over generations. Identification of sources of unwanted variation and quantification of their effect allowed the development of new strategies designed to control spurious variation. Growth and phenotyping of all plants from all generations together, using standard operating procedures (SOP), led to a reduction in extraneous variation associated with transgene expression level quantification. Adoption of such strategies is key to improving the reproducibility of transgenic studies conducted over generations.     

Quantitative GFP fluorescence as an indicator of recombinant protein synthesis in transgenic plants

The utility of green fluorescent protein (GFP) for biological research is evident. A fluorescence-based method was developed to quantify GFP levels in transgenic plants and protein extracts. Fluorescence intensity was linear with increasing levels of GFP over a range that encompasses transgene expression in plants by the cauliflower mosaic virus 35S promoter. Standard curves were used to estimate GFP concentration in planta and in protein extracts. These values were consistent with ELISA measurements of GFP in protein extracts from transgenic plants, indicating that the technique is a reliable measure of recombinant GFP expression. The levels of in planta GFP expression in both homozygous and hemizygous plants was then estimated. Homozygous transgenic plants expressed twice the amount of GFP than hemizygous plants, suggesting additive transgene expression. This methodology may be useful to simplify the characterization of transgene expression in plants.Abbreviations ELISA Enzyme-linked immunosorbent assay - HRP Horseradish peroxidase - GFP Green fluorescent protein Communicated by M.C. Jordan     

Determination of transgene repeat formation and promoter methylation in transgenic plants

The integration of transgenes into a plant host genome following Agrobacterium tumefaciens-mediated or direct transformation may occur as a single copy or in the form of tandem repeats. The latter has been associated with promoter methylation and silencing of transgenes. Thus, the early screening of such transgenic plants is desirable for ruling out future repeat-dependent transgene instability. We developed a simple PCR-based method in which primer pairs were specifically designed so that amplifications could only be obtained if the transgene was present in the form of multiple inserts in a transgenic line. The method was established using 35S-rolC transgenic aspen lines showing morphologically visible transgenic silencing. Later, it was possible to screen independent transgenic lines showing no visible marker gene expression. Furthermore, a method was developed in which positive PCR amplification was indicative of promoter methylation. The results were consistent and reproducible across different independent transgenic lines. The methods were quick, reliable, consistent and reproducible, and can be useful for routine screening of transgene silencing in lines derived from many different systems.     

Transformation of Plants with Multiple Cassettes Generates Simple Transgene Integration Patterns and High Expression Levels

We transformed rice (Oryza sativa L.) simultaneously with five minimal cassettes, each containing a promoter, coding region and polyadenylation site but no vector backbone. We found that multi-transgene cotransformation was achieved with high efficiency using multiple cassettes, with all transgenic plants we generated containing at least two transgenes and 16% containing all five. About 75% of the plants had simple transgene integration patterns with a predominance of single-copy insertions. The expression levels for all transgenes, and the overall coexpression frequencies, were much higher than previously reported in whole plasmid transformants. Four of five lines analyzed for transgene expression stability in subsequent generations showed stable and high expression levels over generations. A simple model is proposed, which accounts for differences in the molecular make-up and the expression profile of transgenic plants generated using whole plasmid or minimal cassettes. We conclude that gene transfer using minimal cassettes is an efficient and rapid method for the production of transgenic plants containing and stably expressing several different transgenes. Our results facilitate effective manipulation of multi-gene pathways in plants in a single transformation step.     

Approaches to Minimize Variation of Transgene Expression in Plants

Genetic transformation of plants has become a widely used technology that serves multiple purposes in plant biology research. However, considerable variation of transgene expression is often observed within populations of transgenic plants transformed with the same transgene construct. This inter-transformant variation of transgene expression hampers proper evaluation of transgenes and might be most undesirable when high-throughput transgene screening is intended. The general plant transformation strategy today is to generate a sufficiently high number of transgenic plants to find some transformants with the desired level of expression. To reduce cost, labor and interpretational flaws, multiple efforts are being directed toward achieving stable expression of transgenes with an expected level of expression. Various factors are thought to contribute to transgene expression variation including the transgene copy number, RNA silencing, transgene insertion site and the employment of certain regulatory sequences to drive transgene expression. This review provides an update on current methodologies to minimize inter-individual variation of transgene expression in nuclear transformed plants.     

TM2, a novel strong matrix attachment region isolated from tobacco, increases transgene expression in transgenic rice calli and plants

Nuclear matrix attachment regions (MARs) are thought to influence the expression of the flanking genes. TM2, a new DNA fragment isolated from tobacco, can bind with the rice nuclear matrix in vitro. In this study, we investigated the effect of TM2 on transgene expression under the control of three different promoters in stably transformed rice calli and plants. The presence of TM2 flanking the transgene increased the expression of constructs based on the constitutive CaMV 35S and maize ubiquitin gene promoters in both resistant calli and transformed plants. The GUS expression directed by the photosynthetic-tissue-specific PNZIP promoter was also increased in photosynthetic tissues of transformants. However, TM2 did not change the gene expression pattern controlled by the PNZIP promoter. The effect of TM2 in transgenic plants was stronger than that in transgenic calli based on all three promoters. Our results indicate that TM2, as a novel strong MAR, can be used to increase the transgene expression levels in the whole plant or in particular tissues of monocotyledons.     

Recurrent Selection for Transgene Activity Levels in Maize Results in Proxy Selection for a Native Gene with the Same Promoter

High activity levels of a transgene can be very useful, making a transgene easier to evaluate for safety and efficacy. High activity levels can also increase the economic benefit of the production of high value proteins in transgenic plants. The goal of this research is to determine if recurrent selection for activity of a transgene will result in higher activity, and if selection for activity of a transgene controlled by a native promoter will also increase protein levels of the native gene with the same promoter. To accomplish this goal we used transgenic maize containing a construct encoding green fluorescent protein controlled by the promoter for the maize endosperm-specific 27kDa gamma zein seed storage protein. We carried out recurrent selection for fluorescence intensity in two breeding populations. After three generations of selection, both selected populations were significantly more fluorescent and had significantly higher levels of 27kDa gamma zein than the unselected control populations. These higher levels of the 27kDa gamma zein occurred independently of the presence of the transgene. The results show that recurrent selection can be used to increase activity of a transgene and that selection for a transgene controlled by a native promoter can increase protein levels of the native gene with the same promoter via proxy selection. Moreover, the increase in native gene protein level is maintained in the absence of the transgene, demonstrating that proxy selection can be used to produce non-transgenic plants with desired changes in gene expression.     

The 35S promoter used in a selectable marker gene of a plant transformation vector affects the expression of the transgene

Positive selection of transgenic plants is essential during plant transformation. Thus, strong promoters are often used in selectable marker genes to ensure successful selection. Many plant transformation vectors, including pPZP family vectors, use the 35S promoter as a regulatory sequence for their selectable marker genes. We found that the 35S promoter used in a selectable marker gene affected the expression pattern of a transgene, possibly leading to a misinterpretation of the result obtained from transgenic plants. It is likely that the 35S enhancer sequence in the 35S promoter is responsible for the interference, as in the activation tagging screen. This affected expression mostly disappeared in transgenic plants generated using vectors without the 35S sequences within their T-DNA region. Therefore, we suggest that caution should be used in selecting a plant transformation vector and in the interpretation of the results obtained from transgenic approaches using vectors carrying the 35S promoter sequences within their T-DNA regions.     

Molecular Characteristics of Transgenic Wheat and the Effect on Transgene Expression

A population of R0 transgenic wheat plants, generated by particle bombardment, was analyzed to define molecular, genetic and phenotypic properties resulting from transformation with a cointegrate vector, or cotransformation with two separate plasmids. By evaluating the progeny of 70 independently-derived transgenic plants, we also identified rare events such as chimerism and transgene elimination, which provide valuable information concerning the development of transgenic cereal plants following bombardment experiments. The frequency of chimerism in our transgenic wheat plants was very low. Furthermore, while transgene elimination did occur, this was also a very rare event. We determined the copy numbers of integrated transgenes and the levels of transgene expression. Comparisons to transgenic rice plants generated in the same manner demonstrated some similarities, but also important differences in transgene behavior. Whereas in rice there is no evidence for any direct relationship between transgene copy number and transgene expression or stability, multicopy populations in wheat demonstrated a bias towards higher levels of expression for the two genes and the maize ubiquitin promoter evaluated in the present study.     

Stringent repression and homogeneous de-repression by tetracycline of a modified CaMV 35S promoter in intact transgenic tobacco plants

A cauliflower mosaic virus (CaMV) 35S promoter derivative, which is tightly repressed by the Tn 10 encoded Tet repressor in a transient expression system as well as in transgenic plants has been constructed. After treatment of transgenic plants with tetracycline (Tc) the activity of the reporter enzyme beta-glucuronidase (GUS) increased up to 500-fold in tissue culture as well as under greenhouse conditions. Efficient de-repression was achieved by Tc uptake through the roots as well as by Tc treatment of leaves of intact plants. As Tc is not very stable in the plants, this system can also be used for a transient expression of a transgene. This system provides a unique tool for regenerating transgenic plants carrying a repressed transgene and for efficiently de-repressing its activity by a specific inducer at any time point of further development.     

羽衣甘蓝胁迫应答基因BoRS1转化烟草的初步研究

将克隆于羽衣甘蓝的胁迫应答基因BoRS1连入中间载体p35S-2300：：gus：：noster相应位点,成功地构建了含BoRS1基因的植物双元表达载体p35S-2300：：BoRS1：：noster,并通过农杆菌介导法对烟草进行了遗传转化。PCR检测结果表明目的基因BoRS1已成功地导入并整合到烟草基因组中。RT-PCR分析显示,在不同的转基因烟草植株中BoRS1表达量存在差异。转BoRS1烟草的耐干性和甘露醇胁迫研究表明,BoRS1基因的表达对提高植物抗干旱胁迫能力有一定的作用。     

Marker gene expression driven by the maize ubiquitin promoter in transgenic wheat

Selecting a promoter for driving transgene expression is one of the most important factors to consider in a transformation project. Information about the native regulation of the promoter activity is important, but it is also necessary to consider how that activity will be affected when integrated into the genome of the transformed plants. Study of a promoter performance in individually transformed lines provides useful information in this area. The maize ubiquitin 1 (Ubi‐1) promoter has been widely used to drive constitutive transgene expression in monocotyledonous plants. However, lack of data on its activity in individual transformed wheat lines constitutes a gap in the understanding and predictability of this promoter's performance. In this paper, we began addressing this problem by examining the expression of the marker gene uidA, coding for β‐glucuronidase (GUS), under the control of the maize Ubi‐1 promoter in individual transgenic wheat (Triticum aestivum L.) lines from different wheat varieties. The expression of uidA driven by this promoter depended to a great extent on the specific transformation event. Whilst expression was strong and constitutive in all tissues in some of the lines analysed, there were also transgenic lines in which GUS activity was restricted to only a few tissues. In general the maize Ubi‐1 promoter had strong activity in young, metabolically active tissues and in pollen grains.