Elevated expression of exogenous Rad51 leads to identical increases in gene-targeting frequency in murine embryonic stem (ES) cells with both functional and dysfunctional p53 genes

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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Production of chimeras by blastocyst and morula injection of targeted ES cells

Gene Targeting ◽

10.1093/oso/9780199637928.003.0008 ◽

1999 ◽

Author(s):

Virginia Papaioannou ◽

Randall Johnson

Keyword(s):

Stem Cells ◽

Gene Targeting ◽

Es Cells ◽

Embryonic Stem ◽

Embryonic Cells ◽

Cell Potential ◽

Teratocarcinoma Cell ◽

Normal Manner ◽

Mammalian Embryos

The ability of mammalian embryos to incorporate foreign cells and develop as chimeras has been exploited for a variety of purposes including the elucidation of cell lineages, the investigation of cell potential, the perpetuation of mutations produced in embryonic stem (ES) cells by gene targeting, and the subsequent analysis of these mutations. The extent of contribution of the foreign cells depends on their developmental synchrony with the host embryo and their mitotic and developmental potential, which may be severely restricted if the cells bear mutations. If the goal in making chimeras is the transmission of a mutation produced by gene targeting to the next generation, the mutant ES cells must have the capacity to undergo meiosis and gametogenesis. Cells from two different mammalian embryos were first combined experimentally to produce a composite animal, dubbed a chimera, nearly four decades ago. Pairs of cleaving, pre-implantation embryos were mechanically associated in vitro until they aggregated together to make single large morulae; these in turn resulted in chimeric offspring (1). Genetic markers were used to distinguish the contributions of the two embryos in these animals. Since then, various methods for making chimeras have been explored to address different types of questions (2). In 1972 it was reported that highly asynchronous embryonic cells, which had been cultured in vitro, could contribute to chimeras upon re-introduction into pre-implantation embryos (3). Not long afterward, several groups working with teratocarcinomas, tumours derived from germ cells of the gonad, discovered that stem cells from these tumours, known as embryonal carcinoma cells, could contribute to an embryo if introduced into pre-implantation stages (4-6). It appeared that the undifferentiated stem cells of the tumour had enough features in common with early embryonic cells that they could respond to the embryonic environment, differentiating in a normal manner, even after long periods in vitro. Their embryonic potential was limited, however, and many teratocarcinoma cell lines made only meagre contributions to the developing fetus or even produced tumours in chimeras (7). Either their derivation from tumours or their extended sojourn in vitro rendered these cells so dissimilar from early embryonic cells that they rarely, if ever, had full embryonic potential.

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Gene Targeting of Cdc42 and Cdc42GAP Affirms the Critical Involvement of Cdc42 in Filopodia Induction, Directed Migration, and Proliferation in Primary Mouse Embryonic Fibroblasts

Molecular Biology of the Cell ◽

10.1091/mbc.e06-05-0466 ◽

2006 ◽

Vol 17 (11) ◽

pp. 4675-4685 ◽

Cited By ~ 111

Author(s):

Linda Yang ◽

Lei Wang ◽

Yi Zheng

Keyword(s):

Gene Targeting ◽

Cell Lines ◽

Cell Cycle Progression ◽

Cell Model ◽

Es Cells ◽

Embryonic Stem ◽

Mouse Embryonic Fibroblasts ◽

Embryonic Fibroblasts ◽

Membrane Ruffling ◽

Primary Mouse

Recent studies in Cdc42 knockout mouse embryonic stem (ES) cells and ES-derived fibroblastoid cell lines raise concern on a body of literature derived by dominant mutant expression approach in a variety of cell lines implicating mammalian Cdc42 as a key regulator of filopodia induction, directional migration and cell cycle progression. To resolve the physiological function of mammalian Cdc42, we have characterized the Cdc42−/− and Cdc42GAP−/− primary mouse embryonic fibroblasts (MEFs) produced by gene targeting as the Cdc42 loss- or gain-of-activity cell model. The Cdc42−/− cells were defective in filopodia formation stimulated by bradykinin and in dorsal membrane ruffling stimulated by PDGF, whereas the Cdc42GAP−/− cells displayed spontaneous filopodia. The Cdc42 loss- or gain-of-activity cells were defective in adhesion to fibronectin, wound-healing, polarity establishment, and migration toward a serum gradient. These defects were associated with deficiencies of PAK1, GSK3β, myosin light chain, and FAK phosphorylation. Furthermore, Cdc42−/− cells were defective in G1/S-phase transition and survival, correlating with deficient NF-κB transcription and defective JNK, p70 S6K, and ERK1/2 activation. These results demonstrate a different requirement of Cdc42 activity in primary MEFs from ES or ES-derived clonal fibroblastoid cells and suggest that Cdc42 plays cell-type–specific signaling roles.

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Analysis of the role of interleukin-1β (IL-1β)in vivo by gene targeting in embryonic stem (ES) cells

Cytokine ◽

10.1016/1043-4666(94)90291-7 ◽

1994 ◽

Vol 6 (5) ◽

pp. 574

Author(s):

L. Shornick ◽

P. De Togni ◽

S. Mariathasan ◽

A. Fick ◽

J. Goellner ◽

...

Keyword(s):

Gene Targeting ◽

Es Cells ◽

Embryonic Stem ◽

Interleukin 1Β

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The length of homology required for gene targeting in embryonic stem cells

Molecular and Cellular Biology ◽

10.1128/mcb.11.11.5586-5591.1991 ◽

1991 ◽

Vol 11 (11) ◽

pp. 5586-5591 ◽

Cited By ~ 1

Author(s):

P Hasty ◽

J Rivera-Pérez ◽

A Bradley

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Homologous Recombination ◽

Gene Targeting ◽

Es Cells ◽

Embryonic Stem ◽

Critical Length ◽

Gene Replacement ◽

Site Specific ◽

The Relationship

Homologous recombination has been used to introduce site-specific mutations into murine embryonic stem (ES) cells with both insertion and replacement vectors. In this study, we compared the frequency of gene targeting with various lengths of homology and found a dramatic increase in targeting with an increase in homology from 1.3 to 6.8 kb. We examined in detail the relationship between the length of homology and the gene-targeting frequency for replacement vectors and found that a critical length of homology is needed for targeting. Adding greater lengths of homology to this critical length has less of an effect on the targeting frequency. We also analyzed the lengths of homology necessary on both arms of the vector for gene replacement events and found that 472 bp of homology is used as efficiently as 1.2 kb in the formation and resolution of crossover junctions.

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A Rapid and Highly Efficient Genotyping Pipeline for Screening Gene Targeted Mouse Embryonic Stem Cells

10.21203/rs.3.rs-748320/v1 ◽

2021 ◽

Author(s):

Roger Caothien ◽

Charles Yu ◽

Lucinda Tam ◽

Robert Newman ◽

Brian Nakao ◽

...

Keyword(s):

Animal Models ◽

Gene Targeting ◽

Fluorescent Protein ◽

Es Cells ◽

Expression Patterns ◽

Embryonic Stem ◽

Rapid Screening ◽

Es Cell ◽

Screening Process ◽

Targeting Vector

Abstract Gene targeting in mouse ES cells replaces or modifies genes of interest; conditional alleles, reporter knock-ins, and amino acid changes are common examples of how gene targeting is used. For example, enhanced green fluorescent protein or Cre recombinase is placed under the control of endogenous genes to define promoter expression patterns. The most important step in the process is to demonstrate that a gene targeting vector is correctly integrated in the genome at the desired chromosomal location. The rapid identification of correctly targeted ES cell clones is facilitated by proper targeting vector construction, rapid screening procedures, and advances in cell culture. The addition of magnetic activated cell sorting (MACS) technology and multiplex droplet digital PCR (ddPCR) to the ES cell screening process can achieve a greater than 60% assurance that ES clones are correctly targeted. In a further refinement of the process, drug selection cassettes are removed from ES cells with adenovirus technology. This improved workflow reduces the time needed to generate preclinical animal models. Faster access to animal models for therapeutic target identification and experimental validation can accelerate the development of therapies for human disease.

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Site-directed point mutations in embryonic stem cells: a gene-targeting tag-and-exchange strategy.

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.4115 ◽

1993 ◽

Vol 13 (7) ◽

pp. 4115-4124 ◽

Cited By ~ 89

Author(s):

G R Askew ◽

T Doetschman ◽

J B Lingrel

Keyword(s):

Gene Targeting ◽

Genomic Sequence ◽

Es Cells ◽

Genetic Disorders ◽

Point Mutations ◽

Embryonic Stem ◽

Gene Cassette ◽

Nucleotide Substitutions ◽

Culture Cells ◽

Atpase Gene

Sequential gene targeting was used to introduce point mutations into one alpha 2 isoform Na,K-ATPase homolog in mouse embryonic stem (ES) cells. In the first round of targeted replacement, the gene was tagged with selectable markers by insertion of a Neor/HSV-tk gene cassette, and this event was selected for by gain of neomycin (G418) resistance. In the second targeted replacement event, the tagged genomic sequence was exchanged with a vector consisting of homologous genomic sequences carrying five site-directed nucleotide substitutions. Embryonic stem cell clones modified by exchange with the mutation vector were selected for loss of the HSV-tk gene by resistance to ganciclovir. Candidate clones were further screened and identified by polymerase chain reaction and Southern blot analysis. By this strategy, the endogenous alpha 2 isoform Na,K-ATPase gene was altered to encode two other amino acids so that the enzyme is resistant to inhibition by cardiac glycosides while maintaining its transmembrane ion-pumping function. Since the initial tagging event and the subsequent mutation-exchange event are independent of one another, a tagged cell line can be used to generate a variety of mutant lines by exchange with various mutation vectors at the tagged locus. This method should be useful for testing specific mutations introduced into the genomes of tissue culture cells and animals and for developing animal models encompassing the mutational variability of known genetic disorders.

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Use of double-replacement gene targeting to replace the murine alpha-lactalbumin gene with its human counterpart in embryonic stem cells and mice.

Molecular and Cellular Biology ◽

10.1128/mcb.14.2.1009 ◽

1994 ◽

Vol 14 (2) ◽

pp. 1009-1016 ◽

Cited By ~ 73

Author(s):

A Stacey ◽

A Schnieke ◽

J McWhir ◽

J Cooper ◽

A Colman ◽

...

Keyword(s):

Gene Targeting ◽

Germ Line ◽

Human Gene ◽

Es Cells ◽

Embryonic Stem ◽

Second Step ◽

Null Mutation ◽

Selection System ◽

Human Counterpart ◽

Alpha Lactalbumin

The mouse alpha-lactalbumin gene has been replaced with the human gene by two consecutive rounds of gene targeting in hypoxanthine phosphoribosyltransferase (HPRT)-deficient feeder-independent murine embryonic stem (ES) cells. One mouse alpha-lactalbumin allele was first replaced by an HPRT minigene which was in turn replaced by human alpha-lactalbumin. The end result is a clean exchange of defined DNA fragments with no other DNA remaining at the target locus. Targeted ES cells at each stage remained capable of contributing efficiently to the germ line of chimeric animals. Double replacement using HPRT-deficient ES cells and the HPRT selection system is therefore a powerful and flexible method of targeting specific alterations to animal genes. A typical strategy for future use would be to generate a null mutation which could then be used to produce multiple second-step alterations at the same locus.

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Genome engineering via homologous recombination in mouse embryonic stem (ES) cells: an amazingly versatile tool for the study of mammalian biology

Anais da Academia Brasileira de Ciências ◽

10.1590/s0001-37652001000300007 ◽

2001 ◽

Vol 73 (3) ◽

pp. 365-383 ◽

Cited By ~ 34

Author(s):

CHARLES BABINET ◽

MICHEL COHEN-TANNOUDJI

Keyword(s):

Homologous Recombination ◽

Gene Targeting ◽

Mouse Genome ◽

Germ Line ◽

Es Cells ◽

Embryonic Stem ◽

Gene Replacement ◽

Knock Out ◽

Genetic Modifications ◽

Cloned Gene

The ability to introduce genetic modifications in the germ line of complex organisms has been a long-standing goal of those who study developmental biology. In this regard, the mouse, a favorite model for the study of the mammals, is unique: indeed not only is it possible since the late seventies, to add genes to the mouse genome like in several other complex organisms but also to perform gene replacement and modification. This has been made possible via two technological breakthroughs: 1) the isolation and culture of embryonic stem cells (ES), which have the unique ability to colonize all the tissues of an host embryo including its germ line; 2) the development of methods allowing homologous recombination between an incoming DNA and its cognate chromosomal sequence (gene ''targeting''). As a result, it has become possible to create mice bearing null mutations in any cloned gene (knock-out mice). Such a possibility has revolutionized the genetic approach of almost all aspects of the biology of the mouse. In recent years, the scope of gene targeting has been widened even more, due to the refinement of the knock-out technology: other types of genetic modifications may now be created, including subtle mutations (point mutations, micro deletions or insertions, etc.) and chromosomal rearrangements such as large deletions, duplications and translocations. Finally, methods have been devised which permit the creation of conditional mutations, allowing the study of gene function throughout the life of an animal, when gene inactivation entails embryonic lethality. In this paper, we present an overview of the methods and scenarios used for the programmed modification of mouse genome, and we underline their enormous interest for the study of mammalian biology.

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Cotransformation and gene targeting in mouse embryonic stem cells

Molecular and Cellular Biology ◽

10.1128/mcb.11.5.2769-2777.1991 ◽

1991 ◽

Vol 11 (5) ◽

pp. 2769-2777

Author(s):

L H Reid ◽

E G Shesely ◽

H S Kim ◽

O Smithies

Keyword(s):

Gene Targeting ◽

Mammalian Cells ◽

Es Cells ◽

Embryonic Stem ◽

Transformed Cells ◽

Neomycin Phosphotransferase ◽

Fold Enrichment ◽

Specific Locus ◽

Dna Fragment ◽

Gene Alterations

We have investigated cotransformation in mammalian cells and its potential for identifying cells that have been modified by gene targeting. Selectable genes on separate DNA fragments were simultaneously introduced into cells by coelectroporation. When the introduced fragments were scored for random integration, 75% of the transformed cells integrated both fragments within the genome of the same cell. When one of the cointroduced fragments was scored for integration at a specific locus by gene targeting, only 4% of the targeted cells cointegrated the second fragment. Apparently, cells that have been modified by gene targeting with one DNA fragment rarely incorporate a second DNA fragment. Despite this limitation, we were able to use the cotransformation protocol to identify targeted cells by screening populations of colonies that had been transformed with a cointroduced selectable gene. When hypoxanthine phosphoribosyltransferase (hprt) targeting DNA was coelectroporated with a selectable neomycin phosphotransferase (neo) gene into embryonic stem (ES) cells, hprt-targeted colonies were isolated from the population of neo transformants at a frequency of 1 per 70 G418-resistant colonies. In parallel experiments with the same targeting construct, hprt-targeted cells were found at a frequency of 1 per 5,500 nonselected colonies. Thus, an 80-fold enrichment for targeted cells was observed within the population of colonies transformed with the cointroduced DNA compared with the population of nonselected colonies. This enrichment for targeted cells after cotransformation should be useful in the isolation of colonies that contain targeted but nonselectable gene alterations.

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