http://www.biomedcentral.com/bmcbiotechnol/ The editor's pick of recent articles published by BMC Biotechnology 2013-04-23T00:00:00Z Selectable marker genes (SMGs) and selection agents are useful tools in the production of transgenic plants by selecting transformed cells from a matrix consisting of of mostly untransformed cells. Most SMGs express protein products that confer antibiotic- or herbicide resistance traits, and typically reside in the end product of genetically-modified (GM) plants. The presence of these genes in GM plants, and subsequently in food, feed and the environment, are of concern and subject to special government regulation in many countries. The presence of SMGs in GM plants might also, in some cases, result in a metabolic burden for the host plants. Their use also prevents the re-use of the same SMG when a second transformation scheme is needed to be performed on the transgenic host. In recent years, several strategies have been developed to remove SMGs from GM products while retaining the transgenes of interest. This review describes the existing strategies for SMG removal, including the implementation of site specific recombination systems, TALENs and ZFNs. This review discusses the advantages and disadvantages of existing SMG-removal strategies and explores possible future research directions for SMG removal including emerging technologies for increased precision for genome modification. http://www.biomedcentral.com/1472-6750/13/36 Yuan-Yeu Yau C Neal Stewart BMC Biotechnology 2013, 13:36 2013-04-23T00:00:00Z 10.1186/1472-6750-13-36 Removing markers from transgenic plants <p>Yuan-Yeu Yau and C Neal Stewart review existing strategies to remove selectable marker genes from transgenic plants, including site specific recombination systems, and explore emerging technologies for increased precision genome modification.</p> /content/figures/1472-6750-13-36-toc.gif BMC Biotechnology 1472-6750 13 36 2013-04-23T00:00:00Z PDF Background: Candidate genes for color pattern formation in butterfly wings have been known based on gene expression patterns since the 1990s, but their functions remain elusive due to a lack of a functional assay. Several methods of transferring and expressing a foreign gene in butterfly wings have been reported, but they have suffered from low success rates or low expression levels. Here, we developed a simple, practical method to efficiently deliver and express a foreign gene using baculovirus-mediated gene transfer in butterfly wings in vivo. Results: A recombinant baculovirus containing a gene for green fluorescent protein (GFP) was injected into pupae of the blue pansy butterfly Junonia orithya (Nymphalidae). GFP fluorescence was detected in the pupal wings and other body parts of the injected individuals three to five days post-injection at various degrees of fluorescence. We obtained a high GFP expression rate at relatively high virus titers, but it was associated with pupal death before color pattern formation in wings. To reduce the high mortality rate caused by the baculovirus treatment, we administered an anti-gp64 antibody, which was raised against baculovirus coat protein gp64, to infected pupae after the baculovirus injection. This treatment greatly reduced the mortality rate of the infected pupae. GFP fluorescence was observed in pupal and adult wings and other body parts of the antibody-treated individuals at various degrees of fluorescence. Importantly, we obtained completely developed wings with a normal color pattern, in which fluorescent signals originated directly from scales or the basal membrane after the removal of scales. GFP fluorescence in wing tissues spatially coincided with anti-GFP antibody staining, confirming that the fluorescent signals originated from the expressed GFP molecules. Conclusions: Our baculovirus-mediated gene transfer system with an anti-gp64 antibody is reasonably efficient, and it can be an invaluable tool to transfer, express, and functionally examine foreign genes in butterfly wings and also in other non-model insect systems. http://www.biomedcentral.com/1472-6750/13/27 Bidur Dhungel Yoshikazu Ohno Rie Matayoshi Joji M Otaki BMC Biotechnology 2013, 13:27 2013-03-25T00:00:00Z 10.1186/1472-6750-13-27 Baculovirus vector delivers foreign gene to butterfly wings <p>A novel baculovirus-mediated gene transfer system is an efficient tool to transfer, express, and functionally examine foreign genes in butterfly wings and may be useful in other non-model insect systems.</p> /content/figures/1472-6750-13-27-toc.gif BMC Biotechnology 1472-6750 13 27 2013-03-25T00:00:00Z XML Background: Tyrosinase is a bifunctional enzyme that catalyzes both the hydroxylation of monophenols to o-diphenols (monophenolase activity) and the subsequent oxidation of the diphenols to o-quinones (diphenolase activity). Due to the potential applications of tyrosinase in biotechnology, in particular in biocatalysis and for biosensors, it is desirable to develop a suitable low-cost process for efficient production of this enzyme. So far, the best production yield reported for tyrosinase was about 1 g L-1, which was achieved by cultivating the filamentous fungus Trichoderma reesei for 6 days. Results: In this work, tyrosinase from Verrucomicrobium spinosum was expressed in Escherichia coli and its production was studied in both batch and fed-batch cultivations. Effects of various key cultivation parameters on tyrosinase production were first examined in batch cultures to identify optimal conditions. It was found that a culture temperature of 32 °C and induction at the late growth stage were favorable, leading to a highest tyrosinase activity of 0.76 U mL-1. The fed-batch process was performed by using an exponential feeding strategy to achieve high cell density. With the fed-batch process, a final biomass concentration of 37 g L-1 (based on optical density) and a tyrosinase activity of 13 U mL-1 were obtained in 28 hours, leading to a yield of active tyrosinase of about 3 g L-1. The highest overall volumetric productivity of 103 mg of active tyrosinase per liter and hour (corresponding to 464 mU L-1 h-1) was determined, which is approximately 15 times higher than that obtained in batch cultures. Conclusions: We have successfully expressed and produced gram quantities per liter of active tyrosinase in recombinant E. coli by optimizing the expression conditions and fed-batch cultivation strategy. Exponential feed of substrate helped to prolong the exponential phase of growth, to reduce the fermentation time and thus the cost. A specific tyrosinase production rate of 103 mg L−1 h−1 and a maximum volumetric activity of 464 mU L−1 h-1 were achieved in this study. These levels have not been reported previously. http://www.biomedcentral.com/1472-6750/13/18 Qun Ren Bernhard Henes Michael Fairhead Linda Thöny-Meyer BMC Biotechnology 2013, 13:18 2013-02-27T00:00:00Z 10.1186/1472-6750-13-18 Optimization of bacterial tyrosinase production <p>Gram quantities per liter of active tyrosinase can be produced in recombinant <em>Escherichia coli</em> by optimizing the expression conditions and utilizing a forward-feed cultivation strategy that reduces fermentation time and thus cost.</p> /content/figures/1472-6750-13-18-toc.gif BMC Biotechnology 1472-6750 13 18 2013-02-27T00:00:00Z XML Background: Transferrin (TF) plays a critical physiological role in cellular iron delivery via the transferrin receptor (TFR)-mediated endocytosis pathway in nearly all eukaryotic organisms. Human serum TF (hTF) is extensively used as an iron-delivery vehicle in various mammalian cell cultures for production of therapeutic proteins, and is also being explored for use as a drug carrier to treat a number of diseases by employing its unique TFR-mediated endocytosis pathway. With the increasing concerns over the risk of transmission of infectious pathogenic agents of human plasma-derived TF, recombinant hTF is preferred to use for these applications. Here, we carry out comparative studies of the TFR binding, TFR-mediated endocytosis and cellular iron delivery of recombinant hTF from rice (rhTF), and evaluate its suitability for biopharmaceutical applications.ResultThrough a TFR competition binding affinity assay with HeLa human cervic carcinoma cells (CCL-2) and Caco-2 human colon carcinoma cells (HTB-37), we show that rhTF competes similarly as hTF to bind TFR, and both the TFR binding capacity and dissociation constant of rhTF are comparable to that of hTF. The endocytosis assay confirms that rhTF behaves similarly as hTF in the slow accumulation in enterocyte-like Caco-2 cells and the rapid recycling pathway in HeLa cells. The pulse-chase assay of rhTF in Caco-2 and HeLa cells further illustrates that rice-derived rhTF possesses the similar endocytosis and intracellular processing compared to hTF. The cell culture assays show that rhTF is functionally similar to hTF in the delivery of iron to two diverse mammalian cell lines, HL-60 human promyelocytic leukemia cells (CCL-240) and murine hybridoma cells derived from a Sp2/0-Ag14 myeloma fusion partner (HB-72), for supporting their proliferation, differentiation, and physiological function of antibody production. Conclusion: The functional similarity between rice derived rhTF and native hTF in their cellular iron delivery, TFR binding, and TFR-mediated endocytosis and intracellular processing support that rice-derived rhTF can be used as a safe and animal-free alternative to serum hTF for bioprocessing and biopharmaceutical applications. http://www.biomedcentral.com/1472-6750/12/92 Deshui Zhang Hsin-Fang Lee Steven C Pettit Jennica L Zaro Ning Huang Wei-Chiang Shen BMC Biotechnology 2012, 12:92 2012-11-30T00:00:00Z 10.1186/1472-6750-12-92 A recombinant alternative to human serum transferrin <p>Rice-derived recombinant human transferrin (rhTF) demonstrates similar transferrin receptor (TFR) binding, TFR-mediated endocytosis and cellular iron delivery function to human transferrin (hTF) and may represent a useful, low-cost alternative for biopharmaceutical applications.</p> /content/figures/1472-6750-12-92-toc.gif BMC Biotechnology 1472-6750 12 92 2012-11-30T00:00:00Z XML