Construction of an Insertion Vector for Gene Targeting of Chicken Lens-specific Gene

The emigration of mature thymocytes from the thymus is critical for establishing peripheral T cell compartments. However, the pathways controlling this process and the timing of egress in relation to postselection developmental stages are poorly defined. Here, we reexamine thymocyte egress and test current and opposing models in relation to the requirement for LTβR, a regulator of thymic microenvironments and thymocyte emigration. Using cell-specific gene targeting, we show that the requirement for LTβR in thymocyte egress is distinct from its control of thymic epithelium and instead maps to expression by endothelial cells. By separating emigration into sequential phases of perivascular space (PVS) entry and transendothelial migration, we reveal a developmentally ordered program of egress where LTβR operates to rate limit access to the PVS. Collectively, we show the process of thymic emigration ensures only the most mature thymocytes leave the thymus and demonstrate a role for LTβR in the initiation of thymus emigration that segregates from its control of medulla organization.

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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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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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