Mutated polyadenylation signals for controlling expression levels of multiple genes in mammalian cells

A set of mutated SV40 early polyadenylation signals (SV40pA) with varying strengths is generated by mutating the AATAAA sequence in the wild-type SV40pA. They are shown to control the expression level of a gene over a 10-fold range using luciferase reporter genes in transient transfection assays. The relative strength of these SV40pA variants remains similar under three commonly used mammalian promoters and in five mammalian cell lines. Application of SV40pA variants for controlling expression level of multiple genes is demonstrated in a study of monoclonal antibody (mAb) synthesis in mammalian cells. By using SV40pA variants of different strengths, the expression of light chain (LC) and heavy chain (HC) genes encoded in a single vector is independently altered which results in different ratios of LC to HC expression spanning a range from 0.24 to 16.42. The changes in gene expression are determined by measuring mRNA levels and intracellular LC and HC polypeptides. It is found that a substantial decrease of HC expression, which increases the LC/HC mRNA ratio, only slightly reduces mAb production. However, reducing the LC expression by a similar magnitude, which decreases the LC/HC mRNA ratio results in a sharp decline of mAb production to trace amounts. This set of SV40pA variants offers a new tool for accurate control of the relative expression levels of multiple genes. It will have wide-ranging applications in fields related to the study of biosynthesis of multi-subunit proteins, proteomic research on protein interactions, and multi-gene metabolic engineering.     

The arcelin-5 Gene of Phaseolus vulgaris Directs High Seed-Specific Expression in Transgenic Phaseolus acutifolius and Arabidopsis Plants

The regulatory sequences of many genes encoding seed storage proteins have been used to drive seed-specific expression of a variety of proteins in transgenic plants. Because the levels at which these transgene-derived proteins accumulate are generally quite low, we investigated the utility of the arcelin-5 regulatory sequences in obtaining high seed-specific expression in transgenic plants. Arcelin-5 is an abundant seed protein found in some wild common bean (Phaseolus vulgaris L.) genotypes. Seeds of Arabidopsis and Tepary bean (Phaseolus acutifolius A. Gray) plants transformed with arcelin-5 gene constructs synthesized arcelin-5 to levels of 15% and 25% of the total protein content, respectively. To our knowledge, such high expression levels directed by a transgene have not been reported before. The transgenic plants also showed low plant-to-plant variation in arcelin expression. Complex transgene integration patterns, which often result in gene silencing effects, were not associated with reduced arcelin-5 expression. High transgene expression was the result of high mRNA steady-state levels and was restricted to seeds. This indicates that all requirements for high seed-specific expression are cis elements present in the cloned genomic arcelin-5 sequence and trans-acting factors that are available in Arabidopsis and Phaseolus spp., and thus probably in most dicotyledonous plants.     

Antisense chalcone synthase genes in petunia: Visualization of variable transgene expression

Summary The constitutive expression of an antisense chalcone synthase (CHS) gene in transgenic petunia plants results with high frequency in a reduced flower pigmentation due to a reduction in the CHS mRNA steady-state level in floral tissue. Here we show that this reduction is specific for CHS mRNA; chalcone flavanone isomerase (CHI) and dihydroflavonol reductase (DFR) mRNA steady-state levels are unaffected. However, in white floral tissue a severe reduction in CHI specific activity is found, accompanied by an altered signal for CHI protein on western blots. We find no correlation between the phenotypic effect of the antisense CHS gene and its chromosomal position. For some of the antisense CHS transformants the flower phenotype is highly variable. We demonstrate that pigmentation in these plants can be influenced by gibberellic acid and light, suggesting that the variable flower phenotype is caused by changes in physiological conditions during flower development. The results not only indicate that flower pigmentation in these plants reveals the variable expression of the antisense transgene, but also show that genomic sequences flanking the transgene may render its expression extremely susceptible to physiological conditions.     

Commercial production of avidin from transgenic maize: characterization of transformant,production, processing,extraction and purification

We have produced in transgenic maize seed the glycoprotein, avidin, which is native to avian, reptilian, and amphibian egg white. A transformant showing high-level expression of avidin was selected. Southern blot data revealed that four copies of the gene are present in this transformant. The foreign protein represents >2% of aqueous soluble extracted protein from populations of dry seed, a level higher than any heterologous protein previously reported for maize. In seed, greater than 55% of the extractable transgenic protein is present in the embryo, an organ representing only 12% of the dry weight of the seed. This indicates that the ubiquitin promoter which is generally considered to be constitutive, in this case may be showing a strong tissue preference in the seed. The mature protein is primarily localized to the intercellular spaces.An interesting trait of the transgenic plants expressing avidin is that the presence of the gene correlates with partial or total male sterility. Seed populations from transgenic plants were maintained by outcrossing and segregate 1:1 for the trait. In generations T2–T4, avidin expression remained high at 2.3% (230 mg/kg seed) of extractable protein from seed, though it varied from 1.5 to 3.0%. However, levels of expression did not appear to depend on pollen parent or growing location. Cracked and flaked kernels stored at –29°C or 10 °C for up to three months showed no significant loss of avidin activity. Commercial processing of harvested seed also generated no apparent loss of activity. The protein was purified to greater than 90% purity by affinity chromatography after extraction from ground mature maize seed. Physical characterization of purified maize-derived avidin demonstrated that the N-terminal amino acid sequence and biotin binding characteristics are identical to the native protein with near identical molecular weight and glycosylation. This study shows that producing avidin from maize is not only possible but has practical advantages over current methods.     

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.     

Expression of the Newcastle disease virus fusion protein in transgenic maize and immunological studies

Transgenic plants have been employed successfully as a low-cost system for the production of therapeutically valuable proteins, including antibodies, antigens and hormones. Here, we report the expression of the fusion (F) gene of the Newcastle disease virus (NDV) in transgenic maize plants. The expression of the transgene, driven by the maize ubiquitin promoter, caused accumulation of the F protein in maize kernels. The presence of the transgene was verified by Southern and western blots. Feeding chickens with kernels containing the F protein induced the production of antibodies, which conferred protection against a viral challenge. This protection was comparable to that conferred by a commercial vaccine. Possible uses of this plant-based F protein as a potential mucosal vaccine are discussed.     

反义trxs基因对转基因小麦种子内源trxh基因表达的影响

以转反义硫氧还蛋白基因(anti-trxs)株系01TY70-1-17-5及其对照小麦品种‘豫麦70’为试验材料,以小麦中稳定表达的肌动蛋白基因actin为内标,用半定量反转录聚合酶链式反应(semi-QRT-PCR)方法,对转基因株系及其对照种子中trxh基因时空表达情况进行了检测。检测结果表明,花后15~30d转基因株系trxh基因转录量平均比对照下降了20.1%,花后25d显著低于对照(P<0.05);胚乳trxh基因转录量最低,平均比对照低19.4%;种子吸涨24h时间内,转基因株系trxh基因转录量较对照均略低,但差异不显著。表明,外源trxs基因的导入直接干扰了内源基因的表达。