PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 14661182

Novel macrolide-adjustable bidirectional expression modules for coordinated expression of two different transgenes in mice.

Cornelia Fux·Wilfried Weber·Marie Daoud-El Baba·Christoph Heinzen·Dominique Aubel·Martin Fussenegger
View original source ↗Export metadata ↓

Abstract

BACKGROUND: Precise control of transgene expression is essential for a variety of applications ranging from gene-function analysis, biopharmaceutical manufacturing to next-generation molecular interventions in gene therapy and tissue engineering. The regulation of gene expression is currently a key issue for clinical implementation of gene-therapy-based treatments since desired transgene expression may need to be maintained within a narrow therapeutic window for successful treatment of a particular human disease. METHODS: We have designed a novel bidirectional expression module that enables adjustable coregulation of two different transgenes in response to clinical doses of macrolide antibiotics. A bidirectional macrolide-responsive promoter consisting of a central operator module (ETR) specific for the macrolide-dependent transactivator (ET1) is flanked by two minimal promoters (P(hCMVmin); P(hsp70min)) which drive expression of two divergently oriented transgenes. Macrolide antibiotics modulate the binding affinity of ET1 to ETR and adjust expression of both transgenes to desired levels. RESULTS: Bidirectional expression configurations enabled excellent macrolide-adjustable coregulation profiles of two secreted reporter genes or one-vector-based autoregulated fine-tuning of a single transgene in various transgenic rodent and human cell lines. Following implantation of microencapsulated CHO-K1 cell derivatives transgenic for macrolide-controlled bidirectional expression of erythropoietin (EPO) and the human secreted alkaline phosphatase (SEAP) intraperitoneally into mice, serum EPO and SEAP levels could be coadjusted to desired levels by administration of different erythromycin doses. CONCLUSIONS: Based on their in vivo compatibility, the versatile bidirectional and macrolide-responsive expression modules represent an important advancement on the way to implementing targeted and conditional molecular interventions into a clinical reality.

Explore related subjects

Alkaline PhosphataseAnimalsCHO CellsCell LineCricetinaeErythropoietinFemaleGene Expression RegulationGenetic EngineeringGenetic TherapyMacrolidesMiceOperator Regions, GeneticPromoter Regions, GeneticTissue EngineeringTransgenesJournal ArticleResearch Support, Non-U.S. Gov't

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Cornelia Fux, Wilfried Weber, Marie Daoud-El Baba, Christoph Heinzen, Dominique Aubel, Martin Fussenegger. 2003. Novel macrolide-adjustable bidirectional expression modules for coordinated expression of two different transgenes in mice.. https://doi.org/10.1002/jgm.443

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2024

A distinct, high-affinity, alkaline phosphatase facilitates occupation of P-depleted environments by marine picocyanobacteria.

Marine picocyanobacteria of the genera Prochlorococcus and Synechococcus, the two most abundant phototrophs on Earth, thrive in oligotrophic oceanic regions. While it is well known that specific lineages are exquisitely adapted to prevailing in situ light and temperature regimes, much less is known of the molecular machinery required to facilitate occupancy of these low-nutrient environments. Here, we describe a hitherto unknown alkaline phosphatase, Psip1, that has a substantially higher affinity for phosphomonoesters than other well-known phosphatases like PhoA, PhoX, or PhoD and is restricted to clade III Synechococcus and a subset of high light I-adapted Prochlorococcus strains, suggesting niche specificity. We demonstrate that Psip1 has undergone convergent evolution with PhoX, requiring both iron and calcium for activity and likely possessing identical key residues around the active site, despite generally very low sequence homology. Interrogation of metagenomes and transcriptomes from TARA oceans and an Atlantic Meridional transect shows that psip1 is abundant and highly expressed in picocyanobacterial populations from the Mediterranean Sea and north Atlantic gyre, regions well recognized to be phosphorus (P)-deplete. Together, this identifies psip1 as an important oligotrophy-specific gene for P recycling in these organisms. Furthermore, psip1 is not restricted to picocyanobacteria and is abundant and highly transcribed in some α-proteobacteria and eukaryotic algae, suggesting that such a high-affinity phosphatase is important across the microbial taxonomic world to occupy low-P environments.

Alkaline Phosphatase↗
PubMed2006

Differentiation of mesenchymal stem cells into osteoblasts on honeycomb collagen scaffolds.

Tissue engineering using living cells is emerging as an alternative to tissue or organ transplantation. The adult mesenchymal stem cells can be differentiated into multilineage cells, such as adipocytes, chondrocytes, or osteoblasts when cultured with specific growth factors. In the present investigation, we have studied the effect of honeycomb collagen scaffolds for the adhesion, differentiation and proliferation of bone marrow-derived mesenchymal stem cells into osteoblasts. Mesenchymal stem cells were isolated from 6-week old albino rat femur bone marrow, and cultured in alpha-MEM medium without beta-glycerophosphate and dexamethasone. Honeycomb collagen discs were prepared from bovine dermal atelocollagen, cross-linked by UV-irradiation and sterilized by heat. The honeycomb discs were placed on the culture dishes before seeding the stem cells. The cells attached quickly to the honeycomb collagen scaffold, differentiated and proliferated into osteoblasts. The differentiated osteoblasts were characterized by morphological examination and alkaline phosphatase activity. The osteoblasts also synthesized calcium-deficient hydroxyapatite (pseudo-hydroxyapatite) crystals in the culture. The mineralization was confirmed by Von Kossa staining and the crystals were analyzed by X-ray diffraction. Light microscopy and DNA measurements showed that the differentiated osteoblasts multiplied into several layers on the honeycomb collagen scaffold. The results demonstrated that the honeycomb collagen sponge is an excellent scaffold for the differentiation and proliferation of mesenchymal stem cells into osteoblasts. The data further proved that honeycomb collagen is an effective substrate for tissue engineering applications, and is very useful in the advancing field of stem cell technology and cell-based therapy.

Alkaline Phosphatase↗
PubMed2006

Leptin effect on RANKL and OPG expression in MC3T3-E1 osteoblasts.

Recent studies have suggested that leptin hormone may play a pivotal role on bone remodeling through a direct effect by modulating positively the OPG/RANKL balance. Here, we investigate the effect of leptin hormone on RANKL and OPG expression in MC3T3-E1 osteoblasts using RT-PCR and ELISA measurements. We have at first identified the expression of Ob-Rb and Ob-Ra leptin receptor isoforms in MC3T3-E1 and observed that these cells respond to mrleptin treatments. We then investigated the effect of mrleptin on RANKL and OPG expression. We show that mrleptin dose-dependently regulated the expression of RANKL mRNA with complete inhibition observed at concentrations higher than 12 ng/ml. This effect was confirmed with sRANKL protein measurements. However, the exposure of MC3T3-E1 to mrleptin had no effect on OPG mRNA. Taken together, these results suggest that leptin modulates positively OPG/RANKL balance by inhibiting the expression of RANKL gene.

Alkaline Phosphatase↗