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5 results on '"Fredrik Sterky"'

1. Efficient Gene-Editing in Human Pluripotent Stem Cells Through Simplified Assembly of Adeno-Associated Viral (AAV) Donor Templates

Author

Berta de La Cruz, Sanhita Mitra, Bingqing He, Melis Çelik, Debora Kaminski, Erik Smedler, and Fredrik Sterky

Subjects
Biology (General), QH301-705.5
Abstract

Gene-edited human pluripotent stem cells provide attractive model systems to functionally interrogate the role of specific genetic variants in relevant cell types. However, the need to isolate and screen edited clones often remains a bottleneck, in particular when recombination rates are sub-optimal. Here, we present a protocol for flexible gene editing combining Cas9 ribonucleoprotein with donor templates delivered by adeno-associated virus (AAV) vectors to yield high rates of homologous recombination. To streamline the workflow, we designed a modular system for one-step assembly of targeting vectors based on Golden Gate cloning and developed a rapid protocol for small-scale isolation of AAV virions of serotype DJ. High homology-directed repair (HDR) rates in human pluripotent stem cells (hPSCs), ~70% in ACTB and ~30% in LMNB1, were achieved using this approach, also with short (300 bp) homology arms. The modular design of donor templates is flexible and allows for the generation of conditional and/or complex alleles. This protocol thus provides a flexible and efficient strategy workflow to rapidly generate gene-edited hPSC lines.

Published
2024
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2. Selection of protein epitopes for antibody production

Author

Mats Lindskog, Johan Rockberg, Mathias Uhlén, and Fredrik Sterky

Subjects
Biology (General), QH301-705.5
Abstract

Protein functional analysis in the post-genomic era is a huge task that has to be approached by different methods in parallel. The use of protein-specific antibodies in conjunction with tissue microarrays has proven to be one important technology. In this study, we present a strategy for the optimized design of protein subfragments for subsequent antibody production. The fragments are selected based on a principle of lowest sequence similarity to other human proteins, optimally to generate antibodies with high selectivity. Furthermore, the fragments should have properties optimized for efficient protein production in Escherichia coli. The strategy has been implemented in Bishop, which is a Java-based software enabling the high-throughput production of protein fragments. Bishop allows for the avoidance of certain restriction enzyme sites, transmembrane regions, and signal peptides. A Basic Local Alignment Search Tool (BLAST) scanning procedure permits the selection of fragments of a selected size with a minimal sequence similarity to other proteins. The software and the strategy were evaluated on a human test data set and verified to fulfill the requested criteria.

Published
2005
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4. The genome of black cottonwood, Populus trichocarpa (Torr. & Gray)

Author

Jérémy Couturier, Astrid Terry, Annabelle Déjardin, Kaisa Nieminen, Chandrashekhar P. Joshi, Wenyu Huang, G.-L. Chen, D. Holligan, Richard A. Jorgensen, Gilles Pilate, Paul G. Richardson, John C. Detter, Uffe Hellsten, Asim Siddiqui, J. Razumovskaya, T. Yin, Francis Martin, Per Unneberg, V. Pereda, Rishikeshi Bhalerao, Carolyn A. Napoli, Jorma Vahala, Jim Leebens-Mack, R. Cunningham, Matias Kirst, Inna Dubchak, Victor Busov, Fredrik Sterky, Daniel S. Rokhsar, Andrew Groover, Sven Degroeve, Marco A. Marra, Alexander Poliakov, Stephane Rombauts, D. Ryaboy, Jean-Charles Leplé, Jaakko Kangasjärvi, Lieven Sterck, Kerr Wall, Pierre Rouzé, Jane Grimwood, N. Islam-Faridi, Michael Gribskov, Jeremy Schmutz, Björn Hamberger, Bo Segerman, Robert Kirkpatrick, Joerg Bohlmann, Damien Blaudez, David R. Nelson, Stefan Jansson, Y. Van de Peer, Sébastien Duplessis, Cécile Rinaldi, Brian E. Ellis, Amy M. Brunner, Robert A. Holt, Chung-Jui Tsai, Sarah F. Covert, Pedro M. Coutinho, Edward C. Uberbacher, Guojun Yang, M. Jones-Rhoades, Udaya C. Kalluri, Carl J. Douglas, J. Schrader, Kermit Ritland, C D Nelson, Bernard Henrissat, Andrea Aerts, Igor V. Grigoriev, Susan R. Wessler, Gerald A. Tuskan, Steven J.M. Jones, Philip F. LoCascio, Barbara Montanini, Yrjö Helariutta, Jacquie Schein, Quentin C. B. Cronk, Lee E. Gunter, H. Shin, Ryan N. Philippe, Ove Nilsson, Göran Sandberg, Susan Lucas, Nicholas H. Putnam, Berthold Heinze, Karla C Gendler, John E. Carlson, Jan Karlsson, Jarrod Chapman, Stephen P. DiFazio, Michel Chalot, Gary F. Peter, Steven G. Ralph, Dawn Cooper, Colin T. Kelleher, Malcolm M. Campbell, Annegret Kohler, Frank W. Larimer, Bill Dirks, Rishikesh P. Bhalerao, John M. Davis, Wout Boerjan, Jürgen Ehlting, Y. Lou, David Goodstein, Annick Brun, Asaf Salamov, Claude W. dePamphilis, Environmental Sciences Division [Oak Ridge], Oak Ridge National Laboratory [Oak Ridge] (ORNL), UT-Battelle, LLC-UT-Battelle, LLC, University of Tennessee, Department of Biology, West Virginia University, Swedish University of Agricultural Sciences (SLU), Michael Smith Laboratories, University of British Columbia (UBC), Joint Genome Institute (JGI), Department of plant systems biology, Flanders Institute for Biotechnology, Canada's Michael Smith Genome Sciences Centre (CMSGSC), BC Cancer Agency (BCCRC), Interactions Arbres-Microorganismes (IAM), Institut National de la Recherche Agronomique (INRA)-Université de Lorraine (UL), Virginia Polytechnic Institute and State University [Blacksburg], Michigan Technological University (MTU), Department of Cell and Systems Biology, University of Toronto, Pennsylvania State University (Penn State), Penn State System, Oak Ridge National Laboratory, Université de la Méditerranée - Aix-Marseille 2, Warnell School of Forest Resources, Department of Botany, University of Florida [Gainesville] (UF), Unité de recherche Amélioration, Génétique et Physiologie Forestières (AGPF), Institut National de la Recherche Agronomique (INRA), University of California [Berkeley], University of California, Genomics Division [LBNL Berkeley], Lawrence Berkeley National Laboratory [Berkeley] (LBNL), University of Arizona, Department of Biological Sciences [Lafayette IN], Purdue University [West Lafayette], Stanford University, US Forest Service, Federal Research and Training Centre for Forests Natural Hazards and Landscape, University of Helsinki, Northern Arizona University [Flagstaff], Department of Plant Biology, Royal Veterinary and Agricultural University = Kongelige Veterinær- og Landbohøjskole (KVL ), United States Department of Agriculture, Texas A&M University System, Whitehead Institute for Biomedical Research and Department of Biology (MIT), Massachusetts Institute of Technology (MIT), Department of Molecular Sciences and Center of Excellence in Genomics and Bioinformatics, The University of Tennessee [Knoxville], Ecologie et Ecophysiologie Forestières [devient SILVA en 2018] (EEF), Department of Forest Sciences [Vancouver], Eberhard Karls Universität Tübingen = Eberhard Karls University of Tuebingen, Royal Institute of Technology in Stockholm (KTH), University of California [Berkeley] (UC Berkeley), University of California (UC), and Helsingin yliopisto = Helsingfors universitet = University of Helsinki

Subjects
0106 biological sciences, Populus trichocarpa, RNA, Untranslated, POPULUS TRICHOCARPA, IDENTIFICATION DE GENES, Arabidopsis, Gene Expression, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Genes, Plant, 01 natural sciences, Genome, Polymorphism, Single Nucleotide, Evolution, Molecular, 03 medical and health sciences, Gene mapping, Gene Duplication, Gene, Populus balsamifera, Phylogeny, 030304 developmental biology, Genomic organization, Oligonucleotide Array Sequence Analysis, Plant Proteins, Genetics, Expressed Sequence Tags, 0303 health sciences, Multidisciplinary, biology, fungi, Chromosome Mapping, Computational Biology, Sequence Analysis, DNA, biology.organism_classification, Protein Structure, Tertiary, Paleopolyploidy, Populus, RNA, Plant, PEUPLIER, Genome, Plant, 010606 plant biology & botany
Abstract

We report the draft genome of the black cottonwood tree, Populus trichocarpa . Integration of shotgun sequence assembly with genetic mapping enabled chromosome-scale reconstruction of the genome. More than 45,000 putative protein-coding genes were identified. Analysis of the assembled genome revealed a whole-genome duplication event; about 8000 pairs of duplicated genes from that event survived in the Populus genome. A second, older duplication event is indistinguishably coincident with the divergence of the Populus and Arabidopsis lineages. Nucleotide substitution, tandem gene duplication, and gross chromosomal rearrangement appear to proceed substantially more slowly in Populus than in Arabidopsis. Populus has more protein-coding genes than Arabidopsis , ranging on average from 1.4 to 1.6 putative Populus homologs for each Arabidopsis gene. However, the relative frequency of protein domains in the two genomes is similar. Overrepresented exceptions in Populus include genes associated with lignocellulosic wall biosynthesis, meristem development, disease resistance, and metabolite transport.

Published
2006
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5. MYB transcription factors are differentially expressed and regulated during secondary vascular tissue development in hybrid aspen.

Author

Barbara Karpinska, Marlene Karlsson, Manoj Srivastava, Anneli Stenberg, Jarmo Schrader, Fredrik Sterky, Rishikesh Bhalerao, and Gunnar Wingsle

Subjects
PLANT cells & tissues, TRANSCRIPTION factors, GENES, TRANSGENIC organisms
Abstract

More than 120,000 poplar ESTs have been sequenced from 20 different cDNA libraries by the Swedish Centre for Tree Functional Genomics. We screened this EST collection for MYB transcription factors involved in secondary vascular tissue formation, and genes assigned as PttMYB3Ra, PttMYB4a and PttMYB21a were selected for further characterisation. Three MYB genes showed different expression patterns in various organs, tissues and stem sub-sections representing different developmental stages of vascular tissue formation. Furthermore, the analysis showed that PttMYB21a expression was much higher in secondary cell wall formation zone of xylem and phloem fibers than in other developmental zones. Transgenic hybrid aspen plants, expressing the 3'-part of the PttMYB21a gene in antisense orientation were generated to assess the function of PttMYB21a gene in vascular tissue formation and lignification. All transgenic lines showed reduced growth and had fewer internodes compared to the wild-type. The analysis of selected lines showed that acid soluble lignin present in the bark was higher in transgenic lines as compared to wild-type plants. Moreover a higher transcript level of caffeoyl-CoA 3-O-methyltransferase [CCoAOMT]; EC 2.1.1.104) was found in the phloem of the transgenic plants, suggesting that PttMYB21a might function as a transcriptional repressor. [ABSTRACT FROM AUTHOR]

Published
2004

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