Springer protocols feed by Genetics/Genomics 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Generation and Characterization of Arabidopsis T-DNA Insertion Mutants
Transfer DNA (T-DNA) insertion mutants are often used in forward and reverse genetics to reveal the molecular mechanisms of a particular biological process in plants. To generate T-DNA insertion mutants, T-DNA must be inserted randomly in the genome through transformation mediated by Agrobacterium tumefaciens. During generation of a T-DNA insertion mutant, Agrobacterium competent cells are first prepared and plasmids containing the T-DNA introduced into Agrobacterium cells. Agrobacterium containing T-DNA vectors are then used to transform T-DNA into Arabidopsis. After screening and identifying T-DNA insertion mutants with ...
Source: Springer protocols feed by Genetics/Genomics - September 26, 2013 Category: Genetics & Stem Cells Source Type: news
Generation and Identification of Arabidopsis EMS Mutants
EMS mutant analysis is a routine experiment to identify new players in a specific biological process or signaling pathway using forward genetics. It begins with the generation of mutants by treating Arabidopsis seeds with EMS. A mutant with a phenotype of interest (mpi) is obtained by screening plants of the M2 generation under a specific condition. Once the phenotype of the mpi is confirmed in the next generation, map-based cloning is performed to locate the mpi mutation. During the map-based cloning, mpi plants (Arabidopsis Columbia-0 (Col-0) ecotype background) are first crossed with Arabidopsis Landsberg erecta (Ler) e...
Source: Springer protocols feed by Genetics/Genomics - September 26, 2013 Category: Genetics & Stem Cells Source Type: news
The Use of Artificial MicroRNA Technology to Control Gene Expression in Arabidopsis thaliana
In plants, double-stranded RNA (dsRNA) is an effective trigger of RNA silencing, and several classes of endogenous small RNA (sRNA), processed from dsRNA substrates by DICER-like (DCL) endonucleases, are essential in controlling gene expression. One such sRNA class, the microRNAs (miRNAs) control the expression of closely related genes to regulate all aspects of plant development, including the determination of leaf shape, leaf polarity, flowering time, and floral identity. A single miRNA sRNA silencing signal is processed from a long precursor transcript of nonprotein-coding RNA, termed the primary miRNA (pri-miRNA). A re...
Source: Springer protocols feed by Genetics/Genomics - September 26, 2013 Category: Genetics & Stem Cells Source Type: news
Tailor-Made Mutations in Arabidopsis Using Zinc Finger Nucleases
Zinc finger nucleases (ZFNs) are proteins engineered to make site-specific double-strand breaks (DSBs) in a DNA sequence of interest. Imprecise repair of the ZFN-induced DSBs by the nonhomologous end-joining (NHEJ) pathway results in a spectrum of mutations, such as nucleotide substitutions, insertions, and deletions. Here we describe a method for targeted mutagenesis in Arabidopsis with ZFNs, which are engineered by context-dependent assembly (CoDA). This ZFN-induced mutagenesis method is an alternative to other currently available gene knockout or knockdown technologies and is useful for reverse genetic studies. (Source:...
Source: Springer protocols feed by Genetics/Genomics - September 26, 2013 Category: Genetics & Stem Cells Source Type: news
Bioinformatic Tools in Arabidopsis Research
Bioinformatic tools are an increasingly important resource for Arabidopsis researchers. With them, it is possible to rapidly query the large data sets covering genomes, transcriptomes, proteomes, epigenomes, and other “omes” that have been generated in the past decade. Often these tools can be used to generate quality hypotheses at the click of a mouse. In this chapter, we cover the use of bioinformatic tools for examining gene expression and coexpression patterns, performing promoter analyses, looking for functional classification enrichment for sets of genes, and investigating protein–protein interactio...
Source: Springer protocols feed by Genetics/Genomics - September 26, 2013 Category: Genetics & Stem Cells Source Type: news
Arabidopsis Database and Stock Resources
The volume of Arabidopsis information has increased enormously in recent years as a result of the sequencing of the reference genome and other large-scale functional genomics projects. Much of the data is stored in public databases, where data are organized, analyzed, and made freely accessible to the research community. These databases are resources that researchers can utilize for making predictions and developing testable hypotheses. The methods in this chapter describe ways to access and utilize Arabidopsis data and genomic resources found in databases and stock centers. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - September 26, 2013 Category: Genetics & Stem Cells Source Type: news
Growing Arabidopsis In Vitro: Cell Suspensions, In Vitro Culture, and Regeneration
An understanding of basic methods in Arabidopsis tissue culture is beneficial for any laboratory working on this model plant. Tissue culture refers to the aseptic growth of cells, organs, or plants in a controlled environment, in which physical, nutrient, and hormonal conditions can all be easily manipulated and monitored. The methodology facilitates the production of a large number of plants that are genetically identical over a relatively short growth period. Techniques, including callus production, cell suspension cultures, and plant regeneration, are all indispensable tools for the study of cellular biochemical and mol...
Source: Springer protocols feed by Genetics/Genomics - September 26, 2013 Category: Genetics & Stem Cells Source Type: news
Using Arabidopsis-Related Model Species (ARMS): Growth, Genetic Transformation, and Comparative Genomics
The Arabidopsis-related model species (ARMS) Thellungiella salsuginea and Thellungiella parvula have generated broad interest in salt stress research. While general growth characteristics of these species are similar to Arabidopsis, some aspects of their life cycle require particular attention in order to obtain healthy plants, with a large production of seeds in a relatively short time. This chapter describes basic procedures for growth, maintenance, and Agrobacterium-mediated transformation of ARMS. Where appropriate, differences in requirements between Thellungiella spp. and Arabidopsis are highlighted, along with basic...
Source: Springer protocols feed by Genetics/Genomics - September 26, 2013 Category: Genetics & Stem Cells Source Type: news
Handling Arabidopsis Plants: Growth, Preservation of Seeds, Transformation, and Genetic Crosses
Growing healthy plants is essential for the advancement of Arabidopsis thaliana (Arabidopsis) research. Over the last 20 years, the Arabidopsis Biological Resource Center (ABRC) has collected and developed a series of best-practice protocols, some of which are presented in this chapter. Arabidopsis can be grown in a variety of locations, growth media, and environmental conditions. Most laboratory accessions and their mutant or transgenic derivatives flower after 4–5 weeks and set seeds after 7–8 weeks, under standard growth conditions (soil, long day, 23 ºC). Some mutant genotypes, natural accessions, and ...
Source: Springer protocols feed by Genetics/Genomics - September 26, 2013 Category: Genetics & Stem Cells Source Type: news
Challenges of “Sticky” Co-immunoprecipitation: Polyalanine Tract Protein–Protein Interactions
Co-immunoprecipitation (Co-IP) (followed by immunoblotting) is a technique widely used to characterize specific protein–protein interactions. Investigating interactions of proteins containing “sticky” polyalanine (PolyA) tracts encounters difficulties using conventional Co-IP procedures. Here, we present strategies to specifically capture proteins containing these difficult PolyA tracts, enabling subsequent robust detection of interacting proteins by Co-IP. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - June 3, 2013 Category: Genetics & Stem Cells Source Type: news
PCR Amplification and Sequence Analysis of GC-Rich Sequences: Aristaless-Related Homeobox Example
PCR amplification (followed by mutation scanning or direct sequencing) is a technique widely used in mutation detection and molecular studies of disease-causing genes, such as ARX. PCR amplification of high GC-rich regions encounters difficulties using conventional PCR procedures. Here, we present the strategies to amplify and sequence these GC-rich regions for the purposes of mutation screening and other molecular analyses. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - June 3, 2013 Category: Genetics & Stem Cells Source Type: news
Characterizing Social Behavior in Genetically Targeted Mouse Models of Brain Disorders
Fragile X syndrome, the leading inherited cause of mental retardation and autism spectrum disorders worldwide, is caused by a tandem repeat expansion in the FMR1 (fragile X mental retardation 1) gene. It presents with a distinct behavioral phenotype which overlaps significantly with that of autism. Emerging evidence suggests that tandem repeat polymorphisms (TRPs) might also play a key role in modulating disease susceptibility for a range of common polygenic disorders, including the broader autism spectrum of disorders (ASD) and other forms of psychiatric illness such as schizophrenia, depression, and bipolar disorder [1]....
Source: Springer protocols feed by Genetics/Genomics - June 3, 2013 Category: Genetics & Stem Cells Source Type: news
Pulse Shape Analysis (PulSA) to Track Protein Translocalization in Cells by Flow Cytometry: Applications for Polyglutamine Aggregation
We describe the basic experimental details for performing PulSA using expression of GFP-tagged proteins, endogenous proteins labelled immunofluorescently, and organelle dyes. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - June 3, 2013 Category: Genetics & Stem Cells Source Type: news
A Method for the Incremental Expansion of Polyglutamine Repeats in Recombinant Proteins
The polyglutamine diseases are caused by the expansion of CAG repeats. A key step in understanding the disease mechanisms, at the DNA and protein level, is the ability to produce recombinant proteins with specific length glutamine tracts which is a time-consuming first step in setting up in vitro systems to study the effects of polyglutamine expansion. Described here is a PCR-based method for the amplification of CAG repeats, which we used to incrementally extend CAG length by 3–5 repeats per cycle. This method could be translated into various contexts where amplification of repeating elements is necessary. (Source: ...
Source: Springer protocols feed by Genetics/Genomics - June 3, 2013 Category: Genetics & Stem Cells Source Type: news
Size Analysis of Polyglutamine Protein Aggregates Using Fluorescence Detection in an Analytical Ultracentrifuge
Defining the aggregation process of proteins formed by poly-amino acid repeats in cells remains a challenging task due to a lack of robust techniques for their isolation and quantitation. Sedimentation velocity methodology using fluorescence detected analytical ultracentrifugation is one approach that can offer significant insight into aggregation formation and kinetics. While this technique has traditionally been used with purified proteins, it is now possible for substantial information to be collected with studies using cell lysates expressing a GFP-tagged protein of interest. In this chapter, we describe protocols for ...
Source: Springer protocols feed by Genetics/Genomics - June 3, 2013 Category: Genetics & Stem Cells Source Type: news
