scholarly journals Efficient gene targeting and removal of foreign DNA by homologous recombination in the picoeukaryoteOstreococcus

2014 ◽  
Vol 78 (6) ◽  
pp. 1073-1083 ◽  
Author(s):  
Jean-Claude Lozano ◽  
Philippe Schatt ◽  
Hugo Botebol ◽  
Valérie Vergé ◽  
Emmanuel Lesuisse ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Efficient Gene ◽  
Foreign Dna

scholarly journals Efficient Gene Targeting by Homologous Recombination in Rat Embryonic Stem Cells

PLoS ONE ◽  
2010 ◽  
Vol 5 (12) ◽  
pp. e14225 ◽  
Author(s):  
Stephen Meek ◽  
Mia Buehr ◽  
Linda Sutherland ◽  
Alison Thomson ◽  
John J. Mullins ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Homologous Recombination ◽  
Gene Targeting ◽  
Embryonic Stem ◽  
Efficient Gene

Is it possible to improve homologous recombination in Chlamydomonas reinhardtii?

Biologia ◽  
2008 ◽  
Vol 63 (6) ◽  
Author(s):  
Miroslava Slaninová ◽  
Dominika Hroššová ◽  
Daniel Vlček ◽  
Wolfgang Mages
Keyword(s):  
Homologous Recombination ◽  
Chlamydomonas Reinhardtii ◽  
Gene Targeting ◽  
Biological Effects ◽  
Model System ◽  
Controlled Environment ◽  
Specific Sequence ◽  
Cellular Processes ◽  
Interesting Object ◽  
Foreign Dna

AbstractTargeted modification of the genome has long been an aim of many geneticists and biotechnologists. Gene targeting is a main molecular tool to examine biological effects of genes in a controlled environment. Effective gene targeting depends on the frequency of homologous recombination that is indispensable for the insertion of foreign DNA into a specific sequence of the genome. The main problem associated with the development of an optimal procedure for gene targeting in a particular organism is the variability of homologous recombination (HR) in different species. Chlamydomonas reinhardtii is an attractive model system for the study of many cellular processes and is also an interesting object for the biotechnology industry. In spite of many advantages of this model system, C. reinhardtii does not readily express heterologous genes and does not allow targeted integration of foreign DNA into its genome easily. This paper compares data obtained from several different experiments designed for improving gene targeting in different organisms and reviews the suitability of particular techniques in C. reinhardtii cells.

scholarly journals Efficient CRISPR/Cas-mediated homologous recombination in the model diatom Thalassiosira pseudonana

2017 ◽  
Author(s):  
Nigel Belshaw ◽  
Irina Grouneva ◽  
Lior Aram ◽  
Assaf Gal ◽  
Amanda Hopes ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Cell Size ◽  
Gene Targeting ◽  
Algal Species ◽  
Thalassiosira Pseudonana ◽  
Cycle Phase ◽  
Metabolic Potential ◽  
Highly Efficient ◽  
Efficient Gene ◽  
Photosynthetic Organism

AbstractCRISPR/Cas enables targeted genome editing in many different plant and algal species including the model diatom Thalassiosira pseudonana. However, efficient gene targeting by homologous recombination (HR) to date is only reported for photosynthetic organisms in their haploid life-cycle phase and there are no examples of efficient nuclease-meditated HR in any photosynthetic organism. Here, a CRISPR/Cas construct, assembled using Golden Gate cloning, enabled highly efficient HR for the first time in a diploid photosynthetic organism. HR was induced in T. pseudonana by means of sequence specific CRISPR/Cas, paired with a donor matrix, generating substitution of the silacidin gene by a resistance cassette (FCP:NAT). Approximately 85% of NAT resistant T. pseudonana colonies screened positive for HR using a nested PCR approach and confirmed by sequencing of the PCR products. The knockout of the silacidin gene in T. pseudonana caused a significant increase in cell size, confirming the role of this gene for cell-size regulation in centric diatoms. Highly efficient gene targeting by HR makes T. pseudonana as genetically tractable as Nannochloropsis and Physcomitrella, hence rapidly advancing functional diatom biology, bionanotechnology and any biotechnological application targeted on harnessing the metabolic potential of diatoms.

Efficient gene targeting by homologous recombination in rice

2002 ◽  
Vol 20 (10) ◽  
pp. 1030-1034 ◽  
Author(s):  
Rie Terada ◽  
Hiroko Urawa ◽  
Yoshishige Inagaki ◽  
Kazuo Tsugane ◽  
Shigeru Iida
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Efficient Gene

Gene Targeting

1999 ◽  
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Mammalian Cells ◽  
Es Cells ◽  
Practical Approach ◽  
Simple Method ◽  
Mouse Es Cells ◽  
Cell Clones ◽  
Previous Edition ◽  
Pros And Cons

Since the publication of the first edition of Gene Targeting: A Practical Approach in 1993 there have been many advances in gene targeting and this new edition has been thoroughly updated and rewritten to include all the major new techniques. It provides not only tried-and-tested practical protocols but detailed guidance on their use and applications. As with the previous edition Gene Targeting: A Practical Approach 2e concentrates on gene targeting in mouse ES cells, but the techniques described can be easily adapted to applications in tissue culture including those for human cells. The first chapter covers the design of gene targeting vectors for mammalian cells and describes how to distinguish random integrations from homologous recombination. It is followed by a chapter on extending conventional gene targeting manipulations by using site-specific recombination using the Cre-loxP and Flp-FRT systems to produce 'clean' germline mutations and conditionally (in)activating genes. Chapter 3 describes methods for introducing DNA into ES cells for homologous recombination, selection and screening procedures for identifying and recovering targeted cell clones, and a simple method for establishing new ES cell lines. Chapter 4 discusses the pros and cons or aggregation versus blastocyst injection to create chimeras, focusing on the technical aspects of generating aggregation chimeras and then describes some of the uses of chimeras. The next topic covered is gene trap strategies; the structure, components, design, and modification of GT vectors, the various types of GT screens, and the molecular analysis of GT integrations. The final chapter explains the use of classical genetics in gene targeting and phenotype interpretation to create mutations and elucidate gene functions. Gene Targeting: A Practical Approach 2e will therefore be of great value to all researchers studying gene function.

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