Publications by authors named "Helene Eckert"

6 Publications

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Strategies for genetic inactivation of long noncoding RNAs in zebrafish.

RNA 2019 08 1;25(8):897-904. Epub 2019 May 1.

Institut Curie, PSL Research University, CNRS UMR3215, INSERM U934, 75005 Paris, France.

The number of annotated long noncoding RNAs (lncRNAs) continues to grow; however, their functional characterization in model organisms has been hampered by the lack of reliable genetic inactivation strategies. While partial or full deletions of lncRNA loci disrupt lncRNA expression, they do not permit the formal association of a phenotype with the encoded transcript. Here, we examined several alternative strategies for generating lncRNA null alleles in zebrafish and found that they often resulted in unpredicted changes to lncRNA expression. Removal of the transcription start sites (TSSs) of lncRNA genes resulted in hypomorphic mutants, due to the usage of either constitutive or tissue-specific alternative TSSs. Deletions of short, highly conserved lncRNA regions can also lead to overexpression of truncated transcripts. In contrast, knock-in of a polyadenylation signal enabled complete inactivation of , the most abundant vertebrate lncRNA. In summary, lncRNA null alleles require extensive in vivo validation, and we propose insertion of transcription termination sequences as the most reliable approach to generate lncRNA-deficient zebrafish.
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http://dx.doi.org/10.1261/rna.069484.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6633201PMC
August 2019

Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR.

Genome Biol 2016 07 5;17(1):148. Epub 2016 Jul 5.

INSERM U1154, CNRS UMR 7196, Muséum National d'Histoire Naturelle, Paris, France.

Background: The success of the CRISPR/Cas9 genome editing technique depends on the choice of the guide RNA sequence, which is facilitated by various websites. Despite the importance and popularity of these algorithms, it is unclear to which extent their predictions are in agreement with actual measurements.

Results: We conduct the first independent evaluation of CRISPR/Cas9 predictions. To this end, we collect data from eight SpCas9 off-target studies and compare them with the sites predicted by popular algorithms. We identify problems in one implementation but found that sequence-based off-target predictions are very reliable, identifying most off-targets with mutation rates superior to 0.1 %, while the number of false positives can be largely reduced with a cutoff on the off-target score. We also evaluate on-target efficiency prediction algorithms against available datasets. The correlation between the predictions and the guide activity varied considerably, especially for zebrafish. Together with novel data from our labs, we find that the optimal on-target efficiency prediction model strongly depends on whether the guide RNA is expressed from a U6 promoter or transcribed in vitro. We further demonstrate that the best predictions can significantly reduce the time spent on guide screening.

Conclusions: To make these guidelines easily accessible to anyone planning a CRISPR genome editing experiment, we built a new website ( http://crispor.org ) that predicts off-targets and helps select and clone efficient guide sequences for more than 120 genomes using different Cas9 proteins and the eight efficiency scoring systems evaluated here.
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http://dx.doi.org/10.1186/s13059-016-1012-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4934014PMC
July 2016

Establishment of a soybean (Glycine max Merr. L) transposon-based mutagenesis repository.

Planta 2009 Jan 15;229(2):279-89. Epub 2008 Oct 15.

National Center for Soybean Biotechnology, Division of Plant Sciences, Life Sciences Center, Department of Molecular Microbiology, University of Missouri, Rollins Road, Columbia, MO 65211, USA.

Soybean is a major crop species providing valuable feedstock for food, feed and biofuel. In recent years, considerable progress has been made in developing genomic resources for soybean, including on-going efforts to sequence the genome. These efforts have identified a large number of soybean genes, most with unknown function. Therefore, a major research priority is determining the function of these genes, especially those involved in agronomic performance and seed traits. One means to study gene function is through mutagenesis and the study of the resulting phenotypes. Transposon-tagging has been used successfully in both model and crop plants to support studies of gene function. In this report, we describe efforts to generate a transposon-based mutant collection of soybean. The Ds transposon system was used to create activation-tagging, gene and enhancer trap elements. Currently, the repository houses approximately 900 soybean events, with flanking sequence data derived from 200 of these events. Analysis of the insertions revealed approximately 70% disrupted known genes, with the majority matching sequences derived from either Glycine max or Medicago truncatula sequences. Among the mutants generated, one resulted in male-sterility and was shown to disrupt the strictosidine synthase gene. This example clearly demonstrates that it is possible to disrupt soybean gene function by insertional mutagenesis and to derive useful mutants by this approach in spite of the tetraploid nature of the soybean genome.
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http://dx.doi.org/10.1007/s00425-008-0827-9DOI Listing
January 2009

Molecular and phenotypic characterization of transgenic soybean expressing the Arabidopsis ferric chelate reductase gene, FRO2.

Planta 2006 Oct 2;224(5):1116-28. Epub 2006 Jun 2.

USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA.

Soybean (Glycine max Merr.) production is reduced under iron-limiting calcareous soils throughout the upper Midwest regions of the US. Like other dicotyledonous plants, soybean responds to iron-limiting environments by induction of an active proton pump, a ferric iron reductase and an iron transporter. Here we demonstrate that heterologous expression of the Arabidopsis thaliana ferric chelate reductase gene, FRO2, in transgenic soybean significantly enhances Fe(+3) reduction in roots and leaves. Root ferric reductase activity was up to tenfold higher in transgenic plants and was not subjected to post-transcriptional regulation. In leaves, reductase activity was threefold higher in the transgenic plants when compared to control. The enhanced ferric reductase activity led to reduced chlorosis, increased chlorophyll concentration and a lessening in biomass loss in the transgenic events between Fe treatments as compared to control plants grown under hydroponics that mimicked Fe-sufficient and Fe-deficient soil environments. However, the data indicate that constitutive FRO2 expression under non-iron stress conditions may lead to a decrease in plant productivity as reflected by reduced biomass accumulation in the transgenic events under non-iron stress conditions. When grown at Fe(III)-EDDHA levels greater than 10 microM, iron concentration in the shoots of transgenic plants was significantly higher than control. The same observation was found in the roots in plants grown at iron levels higher than 32 microM Fe(III)-EDDHA. These results suggest that heterologous expression of an iron chelate reductase in soybean can provide a route to alleviate iron deficiency chlorosis.
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http://dx.doi.org/10.1007/s00425-006-0293-1DOI Listing
October 2006

Co-expression of the borage Delta 6 desaturase and the Arabidopsis Delta 15 desaturase results in high accumulation of stearidonic acid in the seeds of transgenic soybean.

Planta 2006 Oct 23;224(5):1050-7. Epub 2006 May 23.

Department of Agronomy and Horticulture, University of Nebraska-Lincoln, Lincoln, NE 68588-0665, USA.

Two relatively rare fatty acids, gamma-linolenic acid (GLA) and stearidonic acid (STA), have attracted much interest due to their nutraceutical and pharmaceutical potential. STA, in particular, has been considered a valuable alternative source for omega-3 fatty acids due to its enhanced conversion efficiency in animals to eicosapentaenoic acid when compared with the more widely consumed omega-3 fatty acid, alpha-linolenic acid (ALA), present in most vegetable oils. Exploiting the wealth of information currently available on in planta oil biosynthesis and coupling this information with the tool of genetic engineering it is now feasible to deliberately perturb fatty acid pools to generate unique oils in commodity crops. In an attempt to maximize the STA content of soybean oil, a borage Delta(6) desaturase and an Arabidopsis Delta(15) desaturase were pyramided by either sexual crossing of transgenic events, re-transformation of a Delta(6) desaturase event with the Delta(15) desaturase or co-transformation of both desaturases. Expression of both desaturases in this study was under the control of the seed-specific soybean beta-conglycinin promoter. Soybean events that carried only the Delta(15 )desaturase possessed a significant elevation of ALA content, while events with both desaturases displayed a relative STA abundance greater than 29%, creating a soybean with omega-3 fatty acids representing over 60% of the fatty acid profile. Analyses of the membrane lipids in a subset of the transgenic events suggest that soybean seeds compensate for enhanced production of polyunsaturated fatty acids by increasing the relative content of palmitic acid in phosphatidylcholine and other phospholipids.
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http://dx.doi.org/10.1007/s00425-006-0291-3DOI Listing
October 2006