Gene Targeting at the Chromosomal Immunoglobulin Locus: A Model System for the Study of Mammalian Homologous Recombination Mechanisms

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.

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Gene Targeting

10.1093/oso/9780199637928.001.0001 ◽

1999 ◽

Cited By ~ 2

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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Gene conversion in the chicken immunoglobulin locus: A paradigm of homologous recombination in higher eukaryotes

Cellular and Molecular Life Sciences ◽

10.1007/bf01924010 ◽

1994 ◽

Vol 50 (3) ◽

pp. 270-276 ◽

Cited By ~ 19

Author(s):

O. Y. Bezzubova ◽

J. -M. Buerstedde

Keyword(s):

Homologous Recombination ◽

Gene Conversion ◽

Immunoglobulin Locus ◽

Higher Eukaryotes

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Gene replacement with one-sided homologous recombination

Molecular and Cellular Biology ◽

10.1128/mcb.12.1.360-367.1992 ◽

1992 ◽

Vol 12 (1) ◽

pp. 360-367

Author(s):

N Berinstein ◽

N Pennell ◽

C A Ottaway ◽

M J Shulman

Keyword(s):

Homologous Recombination ◽

Dna Sequences ◽

Mammalian Cells ◽

Target Gene ◽

Gene Replacement ◽

Immunoglobulin Genes ◽

Nonhomologous Recombination ◽

Immunoglobulin Locus ◽

Chromosomal Sequences ◽

Dna Vectors

Homologous recombination is now routinely used in mammalian cells to replace endogenous chromosomal sequences with transferred DNA. Vectors for this purpose are traditionally constructed so that the replacement segment is flanked on both sides by DNA sequences which are identical to sequences in the chromosomal target gene. To test the importance of bilateral regions of homology, we measured recombination between transferred and chromosomal immunoglobulin genes when the transferred segment was homologous to the chromosomal gene only on the 3' side. In each of the four recombinants analyzed, the 5' junction was unique, suggesting that it was formed by nonhomologous, i.e., random or illegitimate, recombination. In two of the recombinants, the 3' junction was apparently formed by homologous recombination, while in the other two recombinants, the 3' junction as well as the 5' junction might have involved a nonhomologous crossover. As reported previously, we found that the frequency of gene targeting increases monotonically with the length of the region of homology. Our results also indicate that targeting with fragments bearing one-sided homology can be as efficient as with fragments with bilateral homology, provided that the overall length of homology is comparable. The frequency of these events suggests that the immunoglobulin locus is particularly susceptible to nonhomologous recombination. Vectors designed for one-sided homologous recombination might be advantageous for some applications in genetic engineering.

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

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4509-4517.1991 ◽

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.

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

Scientific Reports ◽

10.1038/srep01532 ◽

2013 ◽

Vol 3 (1) ◽

Cited By ~ 72

Author(s):

Kimitsune Ishizaki ◽

Yasuyo Johzuka-Hisatomi ◽

Sakiko Ishida ◽

Shigeru Iida ◽

Takayuki Kohchi

Keyword(s):

Homologous Recombination ◽

Gene Targeting ◽

Marchantia Polymorpha

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

Eukaryotic Cell ◽

10.1128/ec.00281-14 ◽

2015 ◽

Vol 14 (8) ◽

pp. 783-791 ◽

Cited By ~ 5

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.

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