Molecular biology
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BMC Plant Biology | Full text | Reduced polyphenol oxidase gene expression and enzymatic browning in potato (Solanum tuberosum L.) with artificial microRNAs

Polyphenol oxidase (PPO), often encoded by a multi-gene family, causes oxidative browning, a significant problem in many food products. Low-browning potatoes were produced previously through suppression of PPO gene expression, but the contribution of individual PPO gene isoform to the oxidative browning process was unknown. Here we investigated the contributions of different PPO genes to total PPO protein activity, and the correlations between PPO protein level, PPO activity and tuber tissue browning potential by suppression of all previously characterized potato PPO genes, both individually and in combination using artificial microRNAs (amiRNAs) technology.
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Identified epigenetic factors associated with an increased risk of developing cancer

Identified epigenetic factors associated with an increased risk of developing cancer | Molecular biology | Scoop.it

In 10% of human tumors there is a family history of hereditary disease associated with mutations in identified genes. The best examples are the cases of polyps in the large intestine associated with the APC gene and breast cancer associated with BRCA1 and BRCA2 genes. In the remaining 90% of cases are believed to have an increased risk of developing cancer in relation to genetic variants less powerful but more often, for example, doubles the risk of having a tumor that lacks this small change, called polymorphisms.


Via Integrated DNA Technologies, Jose Eduardo Ulloa Rojas
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Maternal siRNAs as regulators of parental genome imbalance and gene expression in endosperm of Arabidopsis seeds (PNAS)

Maternal siRNAs as regulators of parental genome imbalance and gene expression in endosperm of Arabidopsis seeds (PNAS) | Molecular biology | Scoop.it

Seed size is important to crop domestication and natural selection and is affected by the balance of maternal and paternal genomes in endosperm. Endosperm, like placenta in mammals, provides reserves to the developing embryo. Interploidy crosses disrupt the genome balance in endosperm and alter seed size. Specifically, paternal-excess crosses (2 × 4) delay endosperm cellularization (EC) and produce larger seeds, whereas maternal-excess crosses (4 × 2) promote precocious EC and produce smaller seeds. The mechanisms for responding to the parental genome dosage imbalance and for gene expression changes in endosperm are unknown. In plants, RNA polymerase IV (PolIV or p4) encoded by NRPD1a is required for biogenesis of a major class of 24-nt small interfering RNAs (also known as p4-siRNAs), which are predominately expressed in developing endosperm. Here we show that p4-siRNA accumulation depends on the maternal genome dosage, and maternal p4-siRNAs target transposable elements (TEs) and TE-associated genes (TAGs) in seeds. The p4-siRNAs correlate negatively with expression levels of AGAMOUS-LIKE (AGL) genes in endosperm of interploidy crosses. Moreover, disruption of maternal NRPD1a expression is associated with p4-siRNA reduction and AGL up-regulation in endosperm of reciprocal crosses. This is unique genetic evidence for maternal siRNAs in response to parental genome imbalance and in control of transposons and gene expression during endosperm development.


Via GMI Vienna
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RNA interference (RNAi): by Nature Video

RNA interference (RNAi) is an important process, used by many different organisms to regulate the activity of genes. This animation explains how RNAi works a...

Via Subhas Hajeri
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Metabolic Engineering and Synthetic Biology for the Production of Isoquinoline Alkaloids

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Yit-Lai Chow and Fumihiko Sato

"Plant alkaloids constitute a vast range of compounds with potent bioactivities and are a source of numerous drugs and drug precursors currently used as pharmaceutics. However, the low yield of natural alkaloids in plant tissues limits large-scale drug development. Among the many classes of alkaloids, isoquinoline alkaloids have been intensively studied at the molecular level and efforts to improve their quality and yield are being actively pursued. This chapter describes the major tools used to produce secondary metabolites: (1) synthetic chemistry, (2) metabolic engineering of plants, and (3) synthetic biological approaches. While each of these methods has its advantages and limitations, immense effort in various disciplines as well as combinatorial approaches in metabolic engineering will likely realize an efficient and robust system for the mass production of alkaloids in the foreseeable future."
http://bit.ly/NnSR9x


Via Gerd Moe-Behrens
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A guide to genome engineering with programmable nucleases - Nature Rev. Gen.

A guide to genome engineering with programmable nucleases - Nature Rev. Gen. | Molecular biology | Scoop.it

(via T. Lahaye, thx)

Kim & Kim 2014

Programmable nucleases — including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided engineered nucleases (RGENs) derived from the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)–Cas (CRISPR-associated) system — enable targeted genetic modifications in cultured cells, as well as in whole animals and plants. The value of these enzymes in research, medicine and biotechnology arises from their ability to induce site-specific DNA cleavage in the genome, the repair (through endogenous mechanisms) of which allows high-precision genome editing. However, these nucleases differ in several respects, including their composition, targetable sites, specificities and mutation signatures, among other characteristics. Knowledge of nuclease-specific features, as well as of their pros and cons, is essential for researchers to choose the most appropriate tool for a range of applications.


Via dromius, Jose Eduardo Ulloa Rojas
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Fesquet didier's curator insight, May 28, 2014 8:59 AM

a review of these amazing technique

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True-Breeding Targeted Gene Knock-Out in Barley Using Designer TALE-Nuclease in Haploid Cells - PLOS One

True-Breeding Targeted Gene Knock-Out in Barley Using Designer TALE-Nuclease in Haploid Cells - PLOS One | Molecular biology | Scoop.it

(via T. Lahaye, thx)

Gurushidze et al, 2014

In the present study, we expressed TALENs in pollen-derived, regenerable cells to establish the generation of instantly true-breeding mutant plants. A gfp-specific TALEN pair was expressed via Agrobacterium-mediated transformation in embryogenic pollen of transgenic barley harboring a functional copy of gfp. Thanks to the haploid nature of the target cells, knock-out mutations were readily detected, and homozygous primary mutant plants obtained following genome duplication. In all, 22% of the TALEN transgenics proved knocked out with respect to gfp, and the loss of function could be ascribed to the deletions of between four and 36 nucleotides in length. The altered gfp alleles were transmitted normally through meiosis, and the knock-out phenotype was consistently shown by the offspring of two independent mutants. Thus, here we describe the efficient production of TALEN-mediated gene knock-outs in barley that are instantaneously homozygous and non-chimeric in regard to the site-directed mutations induced. This TALEN approach has broad applicability for both elucidating gene function and tailoring the phenotype of barley and other crop species.


Via dromius, Jose Eduardo Ulloa Rojas
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dromius's curator insight, March 19, 2014 11:01 AM

cool strategy leading to instanteously homozygous plants

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Plant Physiology - Sinauer Associates

Plant Physiology - Sinauer Associates | Molecular biology | Scoop.it
Plant Physiology, Fifth Edition continues to set the standard for textbooks in the field, making plant physiology accessible to virtually every student. Authors Lincoln Taiz and Eduardo Zeiger have again collaborated with a stellar group of co.
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Down-regulation of OsSAG12-1 results in enhanced senescence and pathogen-induced cell death in transgenic rice plants

To evaluate the possible role of OsSAG12-1 we generated RNAi transgenic lines in Japonica rice cultivar TP309. The transgenic lines developed early senescence at varying levels and showed enhanced cell death when inoculated with bacterial pathogen Xanthomonas oryzae pv.oryzae. Our results suggest that OsSAG12-1 is a negative regulator of cell death in rice.


Via Elsa Ballini
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A guide to genome engineering with programmable nucleases - Nature Rev. Gen.

A guide to genome engineering with programmable nucleases - Nature Rev. Gen. | Molecular biology | Scoop.it

(via T. Lahaye, thx)

Kim & Kim 2014

Programmable nucleases — including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided engineered nucleases (RGENs) derived from the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)–Cas (CRISPR-associated) system — enable targeted genetic modifications in cultured cells, as well as in whole animals and plants. The value of these enzymes in research, medicine and biotechnology arises from their ability to induce site-specific DNA cleavage in the genome, the repair (through endogenous mechanisms) of which allows high-precision genome editing. However, these nucleases differ in several respects, including their composition, targetable sites, specificities and mutation signatures, among other characteristics. Knowledge of nuclease-specific features, as well as of their pros and cons, is essential for researchers to choose the most appropriate tool for a range of applications.


Via dromius, Jose Eduardo Ulloa Rojas
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Fesquet didier's curator insight, May 28, 2014 8:59 AM

a review of these amazing technique

Rescooped by Romano Porras from Medical Science
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Enhancing RNA interference with nanoparticles

Enhancing RNA interference with nanoparticles | Molecular biology | Scoop.it
Enhancing RNA interference with nanoparticles

Via Steven Krohn
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Method of the Year 2011: Gene-editing nucleases - by Nature Video - YouTube

Gene-editing nucleases can make targeted and precise changes to an organism's genome. This has opened up new possibilities for the study of gene function, as...
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Top 25 Plasmids of All Time—Addgene Statistics Reveal Research Trends

Top 25 Plasmids of All Time—Addgene Statistics Reveal Research Trends | Molecular biology | Scoop.it

(via T. Lahaye, thx)

a list of the most requested plasmid in each year of Addgene’s history.  Over 800 plasmids in our collection have been distributed over 100 times.  There is amazing diversity in the fields of research covered in these 800 plasmids.  To get a sense of popular research technologies, let’s take a look at our top 25 requested plasmids of all time, listed in the order in which they became available through our service.


Via dromius, Jose Eduardo Ulloa Rojas
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dromius's curator insight, March 28, 2014 4:17 PM
" Our most popular kit of the year was the Golden Gate TALEN Kit, which continues to be our most requested kit of the past 10 years. "
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TALEN utilization in rice genome modifications

TALEN utilization in rice genome modifications | Molecular biology | Scoop.it

Li et al, 2014

Transcription activator-like effector nucleases (TALENs), the newly developed and powerful genetic tools for precise genome editing, are fusion proteins of TAL effectors as DNA binding domains and the cleavage domain of FokI endonuclease. As a pair, the central repeat regions of TALENs determine the DNA binding specificity for the two sub-target sites; and the dimeric non-specific FokI cleavage domains cause a DNA double strand break (DSB) between the bound sequences. In vivo, cells repair the DSBs through either non-homologous end joining (NHEJ) pathway or homologous recombination (HR) pathway. Various methods have been developed for easy and fast assembly of TALEN genes for their utilization in a variety of eukaryotic cells or organisms. Here we present a TALEN-based rice genome modification protocol including constructing modularly assembled TALENs, rice transformation, and mutant screening.


Via dromius, Jose Eduardo Ulloa Rojas
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TALE nickase mediates high efficient targeted transgene integration at the human multi-copy ribosomal DNA locus - Biochem. BioPhys. Res. Comm.

TALE nickase mediates high efficient targeted transgene integration at the human multi-copy ribosomal DNA locus - Biochem. BioPhys. Res. Comm. | Molecular biology | Scoop.it

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Wu et al, 2014

Although targeted gene addition could be stimulated strikingly by a DNA double strand break (DSB) created by either zinc finger nucleases (ZFNs) or TALE nucleases (TALENs), the DSBs are really mutagenic and toxic to human cells. As a compromised solution, DNA single-strand break (SSB) or nick has been reported to mediate high efficient gene addition but with marked reduction of random mutagenesis. We previously demonstrated effective targeted gene addition at the human multicopy ribosomal DNA (rDNA) locus, a genomic safe harbor for the transgene with therapeutic potential. To improve the transgene integration efficiency by using TALENs while lowering the cytotoxicity of DSBs, we created both TALENs and TALE nickases (TALENickases) targeting this multicopy locus. A targeting vector which could integrate a GFP cassette at the rDNA locus was constructed and co-transfected with TALENs or TALENickases. Although the fraction of GFP positive cells using TALENs was greater than that using TALENickases during the first few days after transfection, it reduced to a level less than that using TALENickases after continuous culture. Our findings showed that the TALENickases were more effective than their TALEN counterparts at the multi-copy rDNA locus, though earlier studies using ZFNs and ZFNickases targeting the single-copy loci showed the reverse. Besides, TALENickases mediated the targeted integration of a 5.4 kb fragment at a frequency of up to 0.62% in HT1080 cells after drug selection, suggesting their potential application in targeted gene modification not being limited at the rDNA locus.


Via dromius, Jose Eduardo Ulloa Rojas
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Multiple feedbacks between chloroplast and whole plant in the ...

Abstract. This review focuses on feedback pathways that serve to match plant energy acquisition with plant energy utilization, and thereby aid in the optimization of chloroplast and whole-plant function in a given environment.
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