PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-6 (6)
 

Clipboard (0)
None

Select a Filter Below

Journals
Year of Publication
Document Types
1.  One-Step Agrobacterium Mediated Transformation of Eight Genes Essential for Rhizobium Symbiotic Signaling Using the Novel Binary Vector System pHUGE 
PLoS ONE  2012;7(10):e47885.
Advancement in plant research is becoming impaired by the fact that the transfer of multiple genes is difficult to achieve. Here we present a new binary vector for Agrobacterium tumefaciens mediated transformation, pHUGE-Red, in concert with a cloning strategy suited for the transfer of up to nine genes at once. This vector enables modular cloning of large DNA fragments by employing Gateway technology and contains DsRED1 as visual selection marker. Furthermore, an R/Rs inducible recombination system was included allowing subsequent removal of the selection markers in the newly generated transgenic plants. We show the successful use of pHUGE-Red by transferring eight genes essential for Medicago truncatula to establish a symbiosis with rhizobia bacteria as one 74 kb T-DNA into four non-leguminous species; strawberry, poplar, tomato and tobacco. We provide evidence that all transgenes are expressed in the root tissue of the non-legumes. Visual control during the transformation process and subsequent marker gene removal makes the pHUGE-Red vector an excellent tool for the efficient transfer of multiple genes.
doi:10.1371/journal.pone.0047885
PMCID: PMC3480454  PMID: 23112864
2.  Primer3—new capabilities and interfaces 
Nucleic Acids Research  2012;40(15):e115.
Polymerase chain reaction (PCR) is a basic molecular biology technique with a multiplicity of uses, including deoxyribonucleic acid cloning and sequencing, functional analysis of genes, diagnosis of diseases, genotyping and discovery of genetic variants. Reliable primer design is crucial for successful PCR, and for over a decade, the open-source Primer3 software has been widely used for primer design, often in high-throughput genomics applications. It has also been incorporated into numerous publicly available software packages and web services. During this period, we have greatly expanded Primer3’s functionality. In this article, we describe Primer3’s current capabilities, emphasizing recent improvements. The most notable enhancements incorporate more accurate thermodynamic models in the primer design process, both to improve melting temperature prediction and to reduce the likelihood that primers will form hairpins or dimers. Additional enhancements include more precise control of primer placement—a change motivated partly by opportunities to use whole-genome sequences to improve primer specificity. We also added features to increase ease of use, including the ability to save and re-use parameter settings and the ability to require that individual primers not be used in more than one primer pair. We have made the core code more modular and provided cleaner programming interfaces to further ease integration with other software. These improvements position Primer3 for continued use with genome-scale data in the decade ahead.
doi:10.1093/nar/gks596
PMCID: PMC3424584  PMID: 22730293
3.  Meeting Report from the Second “Minimum Information for Biological and Biomedical Investigations” (MIBBI) workshop 
Standards in Genomic Sciences  2010;3(3):259-266.
This report summarizes the proceedings of the second workshop of the ‘Minimum Information for Biological and Biomedical Investigations’ (MIBBI) consortium held on Dec 1-2, 2010 in Rüdesheim, Germany through the sponsorship of the Beilstein-Institute. MIBBI is an umbrella organization uniting communities developing Minimum Information (MI) checklists to standardize the description of data sets, the workflows by which they were generated and the scientific context for the work. This workshop brought together representatives of more than twenty communities to present the status of their MI checklists and plans for future development. Shared challenges and solutions were identified and the role of MIBBI in MI checklist development was discussed. The meeting featured some thirty presentations, wide-ranging discussions and breakout groups. The top outcomes of the two-day workshop as defined by the participants were: 1) the chance to share best practices and to identify areas of synergy; 2) defining a series of tasks for updating the MIBBI Portal; 3) reemphasizing the need to maintain independent MI checklists for various communities while leveraging common terms and workflow elements contained in multiple checklists; and 4) revision of the concept of the MIBBI Foundry to focus on the creation of a core set of MIBBI modules intended for reuse by individual MI checklist projects while maintaining the integrity of each MI project. Further information about MIBBI and its range of activities can be found at http://mibbi.org/.
doi:10.4056/sigs.147362
PMCID: PMC3035314  PMID: 21304730
4.  RDML: structured language and reporting guidelines for real-time quantitative PCR data 
Nucleic Acids Research  2009;37(7):2065-2069.
The XML-based Real-Time PCR Data Markup Language (RDML) has been developed by the RDML consortium (http://www.rdml.org) to enable straightforward exchange of qPCR data and related information between qPCR instruments and third party data analysis software, between colleagues and collaborators and between experimenters and journals or public repositories. We here also propose data related guidelines as a subset of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to guarantee inclusion of key data information when reporting experimental results.
doi:10.1093/nar/gkp056
PMCID: PMC2673419  PMID: 19223324
5.  Primer3Plus, an enhanced web interface to Primer3 
Nucleic Acids Research  2007;35(Web Server issue):W71-W74.
Here we present Primer3Plus, a new web interface to the popular Primer3 primer design program as an enhanced alternative for the CGI- scripts that come with Primer3. Primer3 consists of a command line program and a web interface. The web interface is one large form showing all of the possible options. This makes the interface powerful, but at the same time confusing for occasional users. Primer3Plus provides an intuitive user interface using present-day web technologies and has been developed in close collaboration with molecular biologists and technicians regularly designing primers. It focuses on the task at hand, and hides detailed settings from the user until these are needed. We also added functionality to automate specific tasks like designing primers for cloning or step-wise sequencing. Settings and designed primer sequences can be stored locally for later use. Primer3Plus supports a range of common sequence formats, such as FASTA. Finally, primers selected by Primer3Plus can be sent to an order form, allowing tight integration into laboratory ordering systems. Moreover, the open architecture of Primer3Plus allows easy expansion or integration of external software packages. The Primer3Plus Perl source code is available under GPL license from SourceForge. Primer3Plus is available at http://www.bioinformatics.nl/primer3plus.
doi:10.1093/nar/gkm306
PMCID: PMC1933133  PMID: 17485472
6.  Liver-Directed Gamma Interferon Gene Delivery in Chronic Hepatitis C 
Journal of Virology  2005;79(21):13412-13420.
Gamma interferon (IFN-γ) has been shown to inhibit replication of subgenomic and genomic hepatitis C virus (HCV) RNAs in vitro and to noncytolytically suppress hepatitis B virus (HBV) replication in vivo. IFN-γ is also known for its immunomodulatory effects and as a marker of a successful cellular immune response to HCV. Therapeutic expression of IFN-γ in the liver may therefore facilitate resolution of chronic hepatitis C, an infection that is rarely resolved spontaneously. To analyze immunomodulatory and antiviral effects of liver-specific IFN-γ expression in vivo, we intravenously injected two persistently HCV-infected chimpanzees twice with a recombinant, replication-deficient HBV vector and subsequently with a recombinant adenoviral vector. These vectors expressed human IFN-γ under control of HBV- and liver-specific promoters, respectively. Gene transfer resulted in a transient increase of intrahepatic IFN-γ mRNA, without increase in serum alanine aminotransferase levels. Ex vivo analysis of peripheral blood lymphocytes demonstrated enhanced CD16 expression on T cells and upregulation of the liver-homing marker CXCR3. Moreover, an increased frequency of HCV-specific T cells was detected ex vivo in the peripheral blood and in vitro in liver biopsy-derived, antigen-nonspecifically expanded T-cell lines. None of these immunologic effects were observed in the third chimpanzee injected with an HBV control vector. Despite these immunologic effects of the experimental vector, however, IFN-γ gene transfer did not result in a significant and long-lasting decrease of HCV titers. In conclusion, liver-directed IFN-γ gene delivery resulted in HCV-specific and nonspecific activation of cellular immune responses but did not result in effective control of HCV replication.
doi:10.1128/JVI.79.21.13412-13420.2005
PMCID: PMC1262601  PMID: 16227262

Results 1-6 (6)