Coordinate expression and independent subcellular targeting of multiple proteins from a single transgene

A variety of conventional methods allow the expression of multiple foreign proteins in plants by transgene stacking or pyramiding. However, most of these approaches have significant drawbacks. We describe a novel alternative, using a single transgene to coordinate expression of multiple proteins that are encoded as a polyprotein capable of dissociating into component proteins on translation. We demonstrate that this polyprotein system is compatible with the need to target proteins to a variety of subcellular locations, either cotranslationally or posttranslationally. It can also be used to coordinate the expression of selectable marker genes and effect genes or to link genes that are difficult to assay to reporter genes that are easily monitored. The unique features of this polyprotein system are based on the novel activity of the 2A peptide of Foot-and-mouth disease virus (FMDV) that acts cotranslationally to effect a dissociation of the polyprotein while allowing translation to continue. This polyprotein system has many applications both as a research tool and for metabolic engineering and protein factory applications of plant biotechnology.     

Use of bicistronic vectors in combination with flow cytometry to screen for effective small interfering RNA target sequences

The efficacy and specificity of small interfering RNAs (siRNAs) are largely dependent on the siRNA sequence. Since only empirical strategies are currently available for predicting these parameters, simple and accurate methods for evaluating siRNAs are needed. To simplify such experiments, target genes are often tagged with reporters for easier readout. Here, we used a bicistronic vector expressing a target gene and green fluorescent protein (GFP) to create a system in which the effect of an siRNA sequence was reflected in the GFP expression level. Cells were transduced with the bicistronic vector, expression vectors for siRNA and red fluorescent protein (RFP). Flow cytometric analysis of the transduced cells revealed that siRNAs for the target gene silenced GFP from the bicistronic vector, but did not silence GFP transcribed without the target gene sequence. In addition, the mean fluorescence intensities of GFP on RFP-expressing cells correlated well with the target gene mRNA and protein levels. These results suggest that this flow cytometry-based method enables us to quantitatively evaluate the efficacy and specificity of siRNAs. Because of its simplicity and effectiveness, this method will facilitate the screening of effective siRNA target sequences, even in high-throughput applications.     

Cell-specific transgene expression from a widely transcribed promoter using Cre/lox in mice

Mice carrying two or more transgenes are used frequently to evaluate oncogene interactions during carcinogenesis. However, neoplastic transformation typically results in reduced expression both of differentiation-specific genes and of transgenes that use their promoters. In contrast, the more widely expressed metallothionein (MT) gene remains expressed at a high level in certain neoplasms, including those developing in pancreas. We have developed a system to maintain high-level, tissue-specific transgene expression during pancreatic carcinogenesis that uses Cre recombinase and a lox site-containing target transgene. Cre was expressed in pancreatic acinar cells under control of the elastase promoter (EL). Cre-mediated target transgene recombination placed a previously silent open-reading frame, encoding rat transforming growth factor alpha (TGFalpha), under control of the MT gene promoter. As long as DNA rearrangement does not occur in other cell types that express MT, TGFalpha expression will be restricted to acinar cells. Development of an effective target transgenic mouse required evaluation of multiple lineages to identify one with sufficient TGFalpha expression to induce pancreatic lesions after transgene rearrangement.     

Autoregulation of transformer-2 alternative splicing is necessary for normal male fertility in Drosophila.

In the male germline of Drosophila the transformer-2 protein is required for differential splicing of pre-mRNAs from the exuperantia and att genes and autoregulates alternative splicing of its own pre-mRNA. Autoregulation of TRA-2 splicing results in production of two mRNAs that differ by the splicing/retention of the M1 intron and encode functionally distinct protein isoforms. Splicing of the intron produces an mRNA encoding TRA-2(226), which is necessary and sufficient for both male fertility and regulation of downstream target RNAs. When the intron is retained, an mRNA is produced encoding TRA-2(179), a protein with no known function. We have previously shown that repression of M1 splicing is dependent on TRA-2(226), suggesting that this protein quantitatively limits its own expression through a negative feedback mechanism at the level of splicing. Here we examine this idea, by testing the effect that variations in the level of tra-2 expression have on the splicing of M1 and on male fertility. Consistent with our hypothesis, we observe that as tra-2 gene dosage is increased, smaller proportions of TRA-2(226) mRNA are produced, limiting expression of this isoform. Feedback regulation is critical for male fertility, since it is significantly decreased by a transgene in which repression of M1 splicing cannot occur and TRA-2(226) mRNA is constitutively produced. The effect of this transgene becomes more severe as its dosage is increased, indicating that fertility is sensitive to an excess of TRA-2(226). Our results suggest that autoregulation of TRA-2(226) expression in male germ cells is necessary for normal spermatogenesis.     

Characterizing standard genetic parts and establishing common principles for engineering legume and cereal roots

Plant synthetic biology and cereal engineering depend on the controlled expression of transgenes of interest. Most engineering in plant species to date has relied heavily on the use of a few, well‐established constitutive promoters to achieve high levels of expression; however, the levels of transgene expression can also be influenced by the use of codon optimization, intron‐mediated enhancement and varying terminator sequences. Most of these alternative approaches for regulating transgene expression have only been tested in small‐scale experiments, typically testing a single gene of interest. It is therefore difficult to interpret the relative importance of these approaches and to design engineering strategies that are likely to succeed in different plant species, particularly if engineering multigenic traits where the expression of each transgene needs to be precisely regulated. Here, we present data on the characterization of 46 promoters and 10 terminators in Medicago truncatula, Lotus japonicus, Nicotiana benthamiana and Hordeum vulgare, as well as the effects of codon optimization and intron‐mediated enhancement on the expression of two transgenes in H. vulgare. We have identified a core set of promoters and terminators of relevance to researchers engineering novel traits in plant roots. In addition, we have shown that combining codon optimization and intron‐mediated enhancement increases transgene expression and protein levels in barley. Based on our study, we recommend a core set of promoters and terminators for broad use and also propose a general set of principles and guidelines for those engineering cereal species.     

Transgenic rice as a system to study the stability of transgene expression: multiple heterologous transgenes show similar behaviour in diverse genetic backgrounds

The success of contemporary breeding programmes involving genetic engineering depends on the stability of transgene expression over many generations. We studied the stability of transgene expression in 40 independent rice plant lines representing 11 diverse cultivated varieties. Each line contained three or four different transgenes delivered by particle bombardment, either by cotransformation or in the form of a cointegrate vector. Approximately 75% of the lines (29/40) demonstrated Mendelian inheritance of all transgenes, suggesting integration at a single locus. We found that levels of transgene expression varied among different lines, but primary transformants showing high-level expression of the gna, gusA, hpt and bar transgenes faithfully transmitted these traits to progeny. Furthermore, we found that cry1Ac and cry2A transgene expression was stably inherited when primary transformants showed moderate or low-level expression. Our results show that six transgenes (three markers and three insect-resistance genes) were stably expressed over four generations of transgenic rice plants. We showed that transgene expression was stable in lines of all the rice genotypes we analysed. Our data represent a step forward in the transfer of rice genetic engineering technology from model varieties to elite breeding lines grown in different parts of the world. Received: 22 March 1999 / Accepted: 6 December 1999