http://www.biomedcentral.com/bmcbiotechnol/ The most accessed research articles published by BMC Biotechnology 2009-11-25T00:00:00Z This is an RSS newsfeed from BioMed Central It is intended to be used with an RSS reader. For more information about RSS newsfeeds from BioMed Central, visit http://www.biomedcentral.com/info/about/rss/ Background: Real-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called 'TaqMan' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive. Results: We have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC) in peripheral blood mononuclear cells); the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy. Conclusion: Our assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting. http://www.biomedcentral.com/1472-6750/3/18 Frederique Ponchel Carmel Toomes Kieran Bransfield Fong Leong Susan Douglas Sandra Bell Valerie Combaret Alain Puisieux Alan Mighell Phill Robinson Chris Inglehearn John Isaacs Alexander Markham Sarah Field BMC Biotechnology 2003, 3:18 2003-10-13T00:00:00Z doi:10.1186/1472-6750-3-18 BMC Biotechnology 1472-6750 3 18 2003-10-13T00:00:00Z XML Background: Global gene expression profiling by DNA microarrays is an invaluable tool in biological research. However, existing labeling methods are time consuming and costly and therefore often limit the scale of microarray experiments and sample throughput. Here we introduce a new, fast, inexpensive method for direct random-primed fluorescent labeling of eukaryotic cDNA for gene expression analysis and compare the results obtained on the NimbleGen microarray platform with two other widely-used labeling methods, namely the NimbleGen-recommended double-stranded cDNA protocol and the indirect (aminoallyl) method. Results: Two total RNA samples were labeled with each method and hybridized to NimbleGen expression arrays. Although all methods tested here provided similar global results and biological conclusions, the new direct random-primed cDNA labeling method provided slightly better correlation between replicates compared to the other methods and thus increased ability to find statistically significant differentially expressed genes. Conclusion: The new direct random-primed cDNA labeling method introduced here is suitable for gene expression microarrays and provides a rapid, inexpensive alternative to existing methods. Using NimbleGen microarrays, the method produced excellent results comparable to those obtained with other methods. However, the simplicity and cost-effectiveness of the new method allows for increased sample throughput in microarray experiments and makes the process amenable to automation with a relatively simple liquid handling system. http://www.biomedcentral.com/1472-6750/9/97 Mario Ouellet Paul Adams Jay Keasling Aindrila Mukhopadhyay BMC Biotechnology 2009, 9:97 2009-11-25T00:00:00Z doi:10.1186/1472-6750-9-97 BMC Biotechnology 1472-6750 9 97 2009-11-25T00:00:00Z PDF Background: Pseudomonas fluorescens is an important food spoilage organism, usually found in the form of biofilms. Bacterial biofilms are inherently resistant to a variety of antimicrobial agents, therefore alternative methods to biofilm control, such as bacteriophages (phages) have been suggested. Phage behavior on biofilms is still poorly investigated and needs further understanding. Here we describe the application of phage ϕIBB-PF7, a newly isolated phage, to control P. fluorescens biofilms. The biofilms were formed under static or dynamic conditions and with or without renewal of medium. Results: Conditions for biofilm formation influenced the feature of the biofilm and the morphology of P. fluorescens. Biomass removal due to phage activity varied between 63 and 91% depending on the biofilm age and the conditions under which the biofilm had been formed and phages applied. Removal of the biofilm by phage treatment was faster in younger biofilms, but the same number of surviving cells was detected in all tested biofilms, after only 4 h of treatment, even in older biofilms. Under static conditions, a 3 log higher number of phage progeny remained either inside the biofilm matrix or attached to the substratum surface than under dynamic conditions, pointing to the importance of experimental conditions for the efficacy of phage entrapment into the biofilm. Conclusion: Phage ϕIBB-PF7A is highly efficient in removing P. fluorescens biofilms within a short time interval. The conditions of biofilm formation and applied during phage infection are critical for the efficacy of the sanitation process. The integration of phages into the biofilm matrix and their entrapment to the surface may be further beneficial factors when phage treatment is considered alone or in addition to chemical biocides in industrial environments where P. fluorescens causes serious spoilage. http://www.biomedcentral.com/1472-6750/8/79 Sanna Sillankorva Peter Neubauer Joana Azeredo BMC Biotechnology 2008, 8:79 2008-10-27T00:00:00Z doi:10.1186/1472-6750-8-79 BMC Biotechnology 1472-6750 8 79 2008-10-27T00:00:00Z XML Background: One theoretical explanation for the relatively poor performance of Brassica rapa (weed) × Brassica napus (crop) transgenic hybrids suggests that hybridization imparts a negative genetic load. Consequently, in hybrids genetic load could overshadow any benefits of fitness enhancing transgenes and become the limiting factor in transgenic hybrid persistence. Two types of genetic load were analyzed in this study: random/linkage-derived genetic load, and directly incorporated genetic load using a transgenic mitigation (TM) strategy. In order to measure the effects of random genetic load, hybrid productivity (seed yield and biomass) was correlated with crop- and weed-specific AFLP genomic markers. This portion of the study was designed to answer whether or not weed × transgenic crop hybrids possessing more crop genes were less competitive than hybrids containing fewer crop genes. The effects of directly incorporated genetic load (TM) were analyzed through transgene persistence data. TM strategies are proposed to decrease transgene persistence if gene flow and subsequent transgene introgression to a wild host were to occur. Results: In the absence of interspecific competition, transgenic weed × crop hybrids benefited from having more crop-specific alleles. There was a positive correlation between performance and number of B. napus crop-specific AFLP markers [seed yield vs. marker number (r = 0.54, P = 0.0003) and vegetative dry biomass vs. marker number (r = 0.44, P = 0.005)]. However under interspecific competition with wheat or more weed-like conditions (i.e. representing a situation where hybrid plants emerge as volunteer weeds in subsequent cropping systems), there was a positive correlation between the number of B. rapa weed-specific AFLP markers and seed yield (r = 0.70, P = 0.0001), although no such correlation was detected for vegetative biomass. When genetic load was directly incorporated into the hybrid genome, by inserting a fitness-mitigating dwarfing gene that that is beneficial for crops but deleterious for weeds (a transgene mitigation measure), there was a dramatic decrease in the number of transgenic hybrid progeny persisting in the population. Conclusion: The effects of genetic load of crop and in some situations, weed alleles might be beneficial under certain environmental conditions. However, when genetic load was directly incorporated into transgenic events, e.g., using a TM construct, the number of transgenic hybrids and persistence in weedy genomic backgrounds was significantly decreased. http://www.biomedcentral.com/1472-6750/9/93 Christy Rose Reginald Millwood Hong Seok Moon Murali Rao Matthew Halfill Paul Raymer Suzanne Warwick Hani Al-Ahmad Jonathan Gressel C Neal Stewart BMC Biotechnology 2009, 9:93 2009-10-31T00:00:00Z doi:10.1186/1472-6750-9-93 BMC Biotechnology 1472-6750 9 93 2009-10-31T00:00:00Z XML Background: Human stem cells are cellular resources with outstanding potential for cell therapy. However, for the fulfillment of this application, major challenges remain to be met. Of paramount importance is the development of robust systems for in vitro stem cell expansion and differentiation. In this work, we successfully developed an efficient scalable bioprocess for the fast production of human neurons. Results: The expansion of undifferentiated human embryonal carcinoma stem cells (NTera2/cl.D1 cell line) as 3D-aggregates was firstly optimized in spinner vessel. The media exchange operation mode with an inoculum concentration of 4 × 105 cell/mL was the most efficient strategy tested, with a 4.6-fold increase in cell concentration achieved in 5 days. These results were validated in a bioreactor where similar profile and metabolic performance were obtained. Furthermore, characterization of the expanded population by immunofluorescence microscopy and flow cytometry showed that NT2 cells maintained their stem cell characteristics along the bioreactor culture time.Finally, the neuronal differentiation step was integrated in the bioreactor process, by addition of retinoic acid when cells were in the middle of the exponential phase. Neurosphere composition was monitored and neuronal differentiation efficiency evaluated along the culture time. The results show that, for bioreactor cultures, we were able to increase significantly the neuronal differentiation efficiency by 10-fold while reducing drastically, by 30%, the time required for the differentiation process. Conclusion: The culture systems developed herein are robust and represent one-step-forward towards the development of integrated bioprocesses, bridging stem cell expansion and differentiation in fully controlled bioreactors. http://www.biomedcentral.com/1472-6750/9/82 Margarida Serra Catarina Brito Eunice Costa Marcos Sousa Paula Alves BMC Biotechnology 2009, 9:82 2009-09-22T00:00:00Z doi:10.1186/1472-6750-9-82 BMC Biotechnology 1472-6750 9 82 2009-09-22T00:00:00Z XML Background: Coccidiosis caused by protozoans of genus Eimeria is a chicken parasitic disease of great economical importance. Conventional disease control strategies depend on vaccination and prophylactic use of anticoccidial drugs. Alternative solution to prevent and treat coccidiosis could be provided by passive immunization using orally delivered neutralizing antibodies. We investigated the possibility to mitigate the parasitic infection by feeding poultry with antibody expressing transgenic crop seeds. Results: Using the phage display antibody library, we generated a panel of anti-Eimeria scFv antibody fragments with high sporozoite-neutralizing activity. These antibodies were expressed either transiently in agrobacteria-infiltrated tobacco leaves or stably in seeds of transgenic pea plants. Comparison of the scFv antibodies purified either from tobacco leaves or from the pea seeds demonstrated no difference in their antigen-binding activity and molecular form compositions. Force-feeding experiments demonstrated that oral delivery of flour prepared from the transgenic pea seeds had higher parasite neutralizing activity in vivo than the purified antibody fragments isolated from tobacco. The pea seed content was found to protect antibodies against degradation by gastrointestinal proteases (>100-fold gain in stability). Ad libitum feeding of chickens demonstrated that the transgenic seeds were well consumed and not shunned. Furthermore, feeding poultry with shred prepared from the antibody expressing pea seeds led to significant mitigation of infection caused both by high and low challenge doses of Eimeria oocysts. Conclusion: The results suggest that our strategy offers a general approach to control parasitic infections in production animals using cost-effective antibody expression in crop seeds affordable for the animal health market. http://www.biomedcentral.com/1472-6750/9/79 Jana Zimmermann Isolde Saalbach Doreen Jahn Martin Giersberg Sigrun Haehnel Julia Wedel Jeanette Macek Karen Zoufal Gerhard Glunder Dieter Falkenburg Sergej Kiprijanov BMC Biotechnology 2009, 9:79 2009-09-11T00:00:00Z doi:10.1186/1472-6750-9-79 BMC Biotechnology 1472-6750 9 79 2009-09-11T00:00:00Z XML Background: A clonal cell line that combines both stable hepatic function and proliferation capacity is desirable for in vitro applications that depend on hepatic function, such as pharmacological or toxicological assays and bioartificial liver systems. Here we describe the generation and characterization of a clonal human cell line for in vitro hepatocyte applications. Results: Cell clones derived from human fetal liver cells were immortalized by over-expression of telomerase reverse transcriptase. The resulting cell line, cBAL111, displayed hepatic functionality similar to the parental cells prior to immortalization, and did not grow in soft agar. Cell line cBAL111 expressed markers of immature hepatocytes, like glutathione S transferase and cytokeratin 19, as well as progenitor cell marker CD146 and was negative for lidocaine elimination. On the other hand, the cBAL111 cells produced urea, albumin and cytokeratin 18 and eliminated galactose. In contrast to hepatic cell lines NKNT-3 and HepG2, all hepatic functions were expressed in cBAL111, although there was considerable variation in their levels compared with primary mature hepatocytes. When transplanted in the spleen of immunodeficient mice, cBAL111 engrafted into the liver and partly differentiated into hepatocytes showing expression of human albumin and carbamoylphosphate synthetase without signs of cell fusion. Conclusion: This novel liver cell line has the potential to differentiate into mature hepatocytes to be used for in vitro hepatocyte applications. http://www.biomedcentral.com/1472-6750/9/89 Tanja Deurholt Niek van Til Aniska Chhatta Lysbeth ten Bloemendaal Ruth Schwartlander Catherine Payne John Plevris Igor Sauer Robert Chamuleau Ronald Oude Elferink Jurgen Seppen Ruurdtje Hoekstra BMC Biotechnology 2009, 9:89 2009-10-21T00:00:00Z doi:10.1186/1472-6750-9-89 BMC Biotechnology 1472-6750 9 89 2009-10-21T00:00:00Z XML Background: When rearing morphologically indistinguishable laboratory strains concurrently, the threat of unintentional genetic contamination is constant. Avoidance of accidental mixing of strains is difficult due to the use of common equipment, technician error, or the possibility of self relocation by adult mosquitoes ("free fliers"). In many cases, laboratory strains are difficult to distinguish because of morphological and genetic similarity, especially when laboratory colonies are isolates of certain traits from the same parental strain, such as eye color mutants, individuals with certain chromosomal arrangements or high levels of insecticide resistance. Thus, proving genetic integrity could seem incredibly time-consuming or impossible. On the other hand, lacking proof of genetically isolated laboratory strains could question the validity of research results. Results: We present a method for establishing authentication matrices to routinely distinguish and confirm that laboratory strains have not become physically or genetically mixed through contamination events in the laboratory. We show a specific example with application to Anopheles gambiae sensu stricto strains at the Malaria Research and Reference Reagent Resource Center. This authentication matrix is essentially a series of tests yielding a strain-specific combination of results. Conclusion: These matrix-based methodologies are useful for several mosquito and insect populations but must be specifically tailored and altered for each laboratory based on the potential contaminants available at any given time. The desired resulting authentication plan would utilize the least amount of routine effort possible while ensuring the integrity of the strains. http://www.biomedcentral.com/1472-6750/9/91 Elien Wilkins Paula Marcet Alice Sutcliffe Paul Howell BMC Biotechnology 2009, 9:91 2009-10-22T00:00:00Z doi:10.1186/1472-6750-9-91 BMC Biotechnology 1472-6750 9 91 2009-10-22T00:00:00Z XML Background: Molecular evolution of carbohydrate binding modules (CBM) is a new approach for the generation of glycan-specific molecular probes. To date, the possibility of performing affinity maturation on CBM has not been investigated. In this study we show that binding characteristics such as affinity can be improved for CBM generated from the CBM4-2 scaffold by using random mutagenesis in combination with phage display technology. Results: Two modified proteins with greatly improved affinity for xyloglucan, a key polysaccharide abundant in the plant kingdom crucial for providing plant support, were generated. Both improved modules differ from other existing xyloglucan probes by binding to galactose-decorated subunits of xyloglucan. The usefulness of the evolved binders was verified by staining of plant sections, where they performed better than the xyloglucan-binding module from which they had been derived. They discriminated non-fucosylated from fucosylated xyloglucan as shown by their ability to stain only the endosperm, rich in non-fucosylated xyloglucan, but not the integument rich in fucosylated xyloglucan, on tamarind seed sections. Conclusion: We conclude that affinity maturation of CBM selected from molecular libraries based on the CBM4-2 scaffold is possible and has the potential to generate new analytical tools for detection of plant carbohydrates. http://www.biomedcentral.com/1472-6750/9/92 Laura von Schantz Fredrika Gullfot Sebastian Scheer Lada Filonova Lavinia Cicortas Gunnarsson James Flint Geoffrey Daniel Eva Nordberg-Karlsson Harry Brumer Mats Ohlin BMC Biotechnology 2009, 9:92 2009-10-31T00:00:00Z doi:10.1186/1472-6750-9-92 BMC Biotechnology 1472-6750 9 92 2009-10-31T00:00:00Z XML Background: Sequencing of the human genome has led to most genes being available in BAC or PAC vectors. However, limited functional information has been assigned to most of these genes. Techniques for the manipulation and transfer of complete functional units on large DNA fragments into human cells are crucial for the analysis of complete genes in their natural genomic context. One limitation of the functional studies using these vectors is the low transfection frequency. Results: We have constructed a shuttle vector, pPAC7, which contains both the EBNA-1 gene and oriP from the Epstein-Barr virus allowing stable maintenance of PAC clones in the nucleus of human cells. The pPAC7 vector also contains the EGFP reporter gene, which allows direct monitoring of the presence of PAC constructs in transfected cells, and the Bsr-cassette that allows highly efficient and rapid selection in mammalian cells by use of blasticidin. Positive selection for recombinant PAC clones is obtained in pPAC7 because the cloning sites are located within the SacBII gene. We show regulated expression of the CDH3 gene carried as a 132 kb genomic insert cloned into pPAC7, demonstrating that the pPAC7 vector can be used for functional studies of genes in their natural genomic context. Furthermore, the results from the transfection of a range of pPAC7 based constructs into two human cell lines suggest that the transfection efficiencies are not only dependent on construct size. Conclusion: The shuttle vector pPAC7 can be used to transfer large genomic constructs into human cells. The genes transferred could potentially contain all long-range regulatory elements, including their endogenous regulatory promoters. Introduction of complete genes in PACs into human cells would potentially allow complementation assays to identify or verify the function of genes affecting cellular phenotypes. http://www.biomedcentral.com/1472-6750/9/88 Hanne Askautrud Elisabet Gjernes Gro Storvold Mona Lindeberg Jim Thorsen Hans Prydz Eirik Frengen BMC Biotechnology 2009, 9:88 2009-10-16T00:00:00Z doi:10.1186/1472-6750-9-88 BMC Biotechnology 1472-6750 9 88 2009-10-16T00:00:00Z XML