Modulating ectopic gene expression levels by using retroviral vectors equipped with synthetic promoters

The human cytomegalovirus and elongation factor 1?? promoters are constitutive promoters commonly employed by mammalian expression vectors. These promoters generally produce high levels of expression in many types of cells and tissues. To generate a library of synthetic promoters capable of generating a range of low, intermediate, and high expression levels, the TATA and CAAT box elements of these promoters were mutated. Other promoter variants were also generated by random mutagenesis. Evaluation using plasmid vectors integrated at a single site in the genome revealed that these various synthetic promoters were capable of expression levels spanning a 40-fold range. Retroviral vectors were equipped with the synthetic promoters and evaluated for their ability to reproduce the graded expression demonstrated by plasmid integration. A vector with a self-inactivating long terminal repeat could neither reproduce the full range of expression levels nor produce stable expression. Using a second vector design, the different synthetic promoters enabled stable expression over a broad range of expression levels in different cell lines.     

Comparative activity of several promoters in driving NIS expression in melanoma cells

Targeted drug delivery systems are special importance for developing gene therapeutic drugs that recognize and eliminate tumor cells. It is desirable that therapeutic genes be expressed predominantly in tumor cells after their targeted delivery into the tumor. Hence, the distribution of the expression product through various tissues should be studied when testing a therapeutic gene in vivo. The sodium iodide symporter (NIS) is attractive as a reporter because its tissue level is easy to quantify by noninvasive imaging methods. Therapeutic gene expression in tumor cells is achieved using various promoters, including strong nonspecific promoters; moderately active tissue-specific promoters; and tumor-specific promoters, which function in a broad range of tumor cells, but have low activity. The relationship between the promoter strength and reporter NIS activity is still unclear. The reporter gene was used to test three promoters types for activity in melanoma cells. The functional activity of NIS expressed from a cloned gene was compared for the three promoters types. Although the promoters greatly varied in strength, only minor changes were observed for NIS functional activity. A relatively weak melanoma-specific promoter ensured a high NIS activity in melanoma cells. Weaker tumorspecific promoters determined a high NIS activity only in some cells of the melanoma origin.     

A portable library of phosphate-depletion based synthetic promoters for customable and automata control of gene expression in bacteria

Industrial biotechnology gene expression systems relay on constitutive promoters compromising cellular growth from the start of the bioprocess, or on inducible devices, which require manual addition of cognate inducers. To overcome this shortcoming, we engineered an automata regulatory system based on cell-stress mechanisms. Specifically, we engineered a synthetic and highly portable phosphate-depletion library of promoters inspired by bacterial PHO starvation system (Pliar promoters). Furthermore, we fully characterized 10 synthetic promoters within the background of two well-known bacterial workhorses such as E. coli W and P. putida KT2440. The promoters displayed an interesting host-dependent performance and a wide strength spectrum ranging from 0.4- to 1.3-fold when compared to the wild-type phosphatase alkaline promoter (PphoA). By comparing with available gene expression systems, we proved the suitability of this new library for the automata and effective decoupling of growth from production in P. putida. Growth phase-dependent expression of these promoters could therefore be activated by fine tuning the initial concentration of phosphate in the medium. Finally, the Pliar library was implemented in the SEVA platform in a ready-to-use mode allowing its broad use by the scientific community.     

Seed-specific promoters direct gene expression in non-seed tissue

The organ specificity of four promoters that are known to direct seed-specific gene expression was tested. Whereas the phaseolin (phas)- and legumin B4 (leB4)-promoters were from genes encoding 7S and 11S globulins from Phaseolus vulgaris and Vicia faba, respectively, the usp- and the sbp-promoters were from non-storage protein genes of V. faba. The expression of different promoter-reporter gene fusions was followed either by RT-PCR or by registering the reporter enzyme activity in organs of transgenic tobacco, pea, narbon bean, or linseed. In addition to seeds, the promoters directed reporter gene expression in pollen and in seed coats. USP-, vicilin- and legumin-mRNA were detected by RT-PCR in pollen of Pisum sativum and V. faba. Expression during microsporogenesis and embryogenesis seems to be a general character of various seed protein genes.     

Transient gene expression and influence of promoters on foreign gene expression in Arabidopsis thaliana

Summary The influence of a variety of parameters was investigated on polyethylene glycol (PEG)-mediated transient nptII and gus gene expression in mesophyll protoplasts of Arabidopsis thaliana ecotype, Estland, in order to develop a suitable transient gene expression system. The investigation revealed that a combination of 20% PEG, incubation time of 15 min, 20–30 μg plasmid concentration per ml along with 50 μg carrier DNA m/l, and inclusion of calcium and magnesium ions during transfection followed by a culture period of 24 h registered maximum NPTII activity. Of the various promoters used for driving expression of the gus gene, the ubiquitin promoter from A. thaliana was the most efficient followed by 35S promoter of the CaMV and the actin promoter of rice. For comparison, similar studies in protoplasts of rice, wheat, and Brassica also revealed the differences in strength of these promoters. Arabidopsis ubiquitin promoter was the most effective in Brassica, and the rice actin1 promoter was the most effective in rice and wheat.     

Recent advances in gene expression data clustering: a case study with comparative results

Several advanced techniques have been proposed for data clustering and many of them have been applied to gene expression data, with partial success. The high dimensionality and the multitude of admissible perspectives for data analysis of gene expression require additional computational resources, such as hierarchical structures and dynamic allocation of resources. We present an immune-inspired hierarchical clustering device, called hierarchical artificial immune network (HaiNet), especially devoted to the analysis of gene expression data. This technique was applied to a newly generated data set, involving maize plants exposed to different aluminum concentrations. The performance of the algorithm was compared with that of a self-organizing map, which is commonly adopted to deal with gene expression data sets. More consistent and informative results were obtained with HaiNet.