scholarly journals Gene Editing a Constitutively Active OsRac1 by Homologous Recombination-Based Gene Targeting Induces Immune Responses in Rice

2013 ◽  
Vol 54 (12) ◽  
pp. 2058-2070 ◽  
Author(s):  
Thu Thi Dang ◽  
Zenpei Shimatani ◽  
Yoji Kawano ◽  
Rie Terada ◽  
Ko Shimamoto
Keyword(s):  
Homologous Recombination ◽  
Immune Responses ◽  
Gene Targeting ◽  
Gene Editing ◽  
Constitutively Active

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.

scholarly journals Unsuccessful attempt at gene-editing by homologous recombination in the zebrafish germ line using the approach of “Rong and Golic”

2012 ◽  
Vol 21 (5) ◽  
pp. 1125-1136 ◽  
Author(s):  
Rosalind Brookfield ◽  
Felix Dafhnis-Calas ◽  
Zhengyao Xu ◽  
William Brown
Keyword(s):  
Homologous Recombination ◽  
Gene Editing ◽  
Germ Line ◽  
Unsuccessful Attempt

scholarly journals Target frequency and integration pattern for insertion and replacement vectors in embryonic stem cells

1991 ◽  
Vol 11 (9) ◽  
pp. 4509-4517
Author(s):  
P Hasty ◽  
J Rivera-Pérez ◽  
C Chang ◽  
A Bradley
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Mammalian Cells ◽  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Homologous Sequence ◽  
Reciprocal Recombination ◽  
Replacement Vector ◽  
Insertion Vector

Gene targeting has been used to direct mutations into specific chromosomal loci in murine embryonic stem (ES) cells. The altered locus can be studied in vivo with chimeras and, if the mutated cells contribute to the germ line, in their offspring. Although homologous recombination is the basis for the widely used gene targeting techniques, to date, the mechanism of homologous recombination between a vector and the chromosomal target in mammalian cells is essentially unknown. Here we look at the nature of gene targeting in ES cells by comparing an insertion vector with replacement vectors that target hprt. We found that the insertion vector targeted up to ninefold more frequently than a replacement vector with the same length of homologous sequence. We also observed that the majority of clones targeted with replacement vectors did not recombine as predicted. Analysis of the recombinant structures showed that the external heterologous sequences were often incorporated into the target locus. This observation can be explained by either single reciprocal recombination (vector insertion) of a recircularized vector or double reciprocal recombination/gene conversion (gene replacement) of a vector concatemer. Thus, single reciprocal recombination of an insertion vector occurs 92-fold more frequently than double reciprocal recombination of a replacement vector with crossover junctions on both the long and short arms.

scholarly journals Homologous recombination-mediated gene targeting in the liverwort Marchantia polymorpha L.

2013 ◽  
Vol 3 (1) ◽  
Author(s):  
Kimitsune Ishizaki ◽  
Yasuyo Johzuka-Hisatomi ◽  
Sakiko Ishida ◽  
Shigeru Iida ◽  
Takayuki Kohchi
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Marchantia Polymorpha

scholarly journals Deletion of the DNA Ligase IV Gene in Candida glabrata Significantly Increases Gene-Targeting Efficiency

2015 ◽  
Vol 14 (8) ◽  
pp. 783-791 ◽  
Author(s):  
Yuke Cen ◽  
Alessandro Fiori ◽  
Patrick Van Dijck
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Candida Glabrata ◽  
The United States ◽  
Genetic Alterations ◽  
Nonhomologous End Joining ◽  
Specific Gene ◽  
Phylogenetic Distance ◽  
End Joining ◽  
Content Type

ABSTRACTCandida glabratais reported as the second most prevalent human opportunistic fungal pathogen in the United States. Over the last decades, its incidence increased, whereas that ofCandida albicansdecreased slightly. One of the main reasons for this shift is attributed to the inherent tolerance ofC. glabratatoward the commonly used azole antifungal drugs. Despite a close phylogenetic distance toSaccharomyces cerevisiae, homologous recombination works with poor efficiency inC. glabratacompared to baker's yeast, in fact limiting targeted genetic alterations of the pathogen's genome. It has been shown that nonhomologous DNA end joining is dominant over specific gene targeting inC. glabrata. To improve the homologous recombination efficiency, we have generated a strain in which theLIG4gene has been deleted, which resulted in a significant increase in correct gene targeting. The very specific function of Lig4 in mediating nonhomologous end joining is the reason for the absence of clear side effects, some of which affect theku80mutant, another mutant with reduced nonhomologous end joining. We also generated aLIG4reintegration cassette. Our results show that thelig4mutant strain may be a valuable tool for theC. glabrataresearch community.

scholarly journals Rational design of antisense oligonucleotides modulating the activity of TLR7/8 agonists

2020 ◽  
Author(s):  
Arwaf S Alharbi ◽  
Aurélie J Garcin ◽  
Kim A Lennox ◽  
Solène Pradeloux ◽  
Christophe Wong ◽  
...  
Keyword(s):  
Immune Responses ◽  
Gene Targeting ◽  
Immune Suppression ◽  
Immune Cells ◽  
Antisense Oligonucleotides ◽  
Rational Design ◽  
Locked Nucleic Acid ◽  
Dependent Manner ◽  
Therapeutic Development ◽  
Proof Of Principle

Abstract Oligonucleotide-based therapeutics have become a reality, and are set to transform management of many diseases. Nevertheless, the modulatory activities of these molecules on immune responses remain incompletely defined. Here, we show that gene targeting 2′-O-methyl (2′OMe) gapmer antisense oligonucleotides (ASOs) can have opposing activities on Toll-Like Receptors 7 and 8 (TLR7/8), leading to divergent suppression of TLR7 and activation of TLR8, in a sequence-dependent manner. Surprisingly, TLR8 potentiation by the gapmer ASOs was blunted by locked nucleic acid (LNA) and 2′-methoxyethyl (2′MOE) modifications. Through a screen of 192 2′OMe ASOs and sequence mutants, we characterized the structural and sequence determinants of these activities. Importantly, we identified core motifs preventing the immunosuppressive activities of 2′OMe ASOs on TLR7. Based on these observations, we designed oligonucleotides strongly potentiating TLR8 sensing of Resiquimod, which preserve TLR7 function, and promote strong activation of phagocytes and immune cells. We also provide proof-of-principle data that gene-targeting ASOs can be selected to synergize with TLR8 agonists currently under investigation as immunotherapies, and show that rational ASO selection can be used to prevent unintended immune suppression of TLR7. Taken together, our work characterizes the immumodulatory effects of ASOs to advance their therapeutic development.

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