Cotransformation and gene targeting in mouse embryonic stem cells

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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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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Gene targeting, principles, and practice in mammalian cells

Gene Targeting ◽

10.1093/oso/9780199637928.003.0005 ◽

1999 ◽

Author(s):

Paul Hasty ◽

Alejandro Abuin

Keyword(s):

Gene Targeting ◽

Mammalian Cells ◽

Es Cells ◽

Globin Gene ◽

Marker Gene ◽

Embryonic Stem ◽

Fibroblast Cell ◽

Chromosomal Locus ◽

Target Locus

When a fragment of genomic DNA is introduced into a mammalian cell it can locate and recombine with the endogenous homologous sequences. This type of homologous recombination, known as gene targeting, is the subject of this chapter. Gene targeting has been widely used, particularly in mouse embryonic stem (ES) cells, to make a variety of mutations in many different loci so that the phenotypic consequences of specific genetic modifications can be assessed in the organism. The first experimental evidence for the occurrence of gene targeting in mammalian cells was made using a fibroblast cell line with a selectable artificial locus by Lin et al. (1), and was subsequently demonstrated to occur at the endogenous β-globin gene by Smithies et al. in erythroleukaemia cells (2). In general, the frequencies of gene targeting in mammalian cells are relatively low compared to yeast cells and this is probably related to, at least in part, a competing pathway: efficient integration of the transfected DNA into a random chromosomal site. The relative ratio of targeted to random integration events will determine the ease with which targeted clones are identified in a gene targeting experiment. This chapter details aspects of vector design which can determine the efficiency of recombination, the type of mutation that may be generated in the target locus, as well as the selection and screening strategies which can be used to identify clones of ES cells with the desired targeted modification. Since the most common experimental strategy is to ablate the function of a target gene (null allele) by introducing a selectable marker gene, we initially describe the vectors and the selection schemes which are helpful in the identification of recombinant clones (Sections 2-5). In Section 6, we describe the vectors and additional considerations for generating subtle mutations in a target locus devoid of any exogenous sequences. Finally, Section 7 is dedicated to the use of gene targeting as a method to express exogenous genes from specific endogenous regulatory elements in vivo, also known as ‘knock-in’ strategies. A targeting vector is designed to recombine with and mutate a specific chromosomal locus.

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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 ◽

1991 ◽

Vol 11 (9) ◽

pp. 4509-4517 ◽

Cited By ~ 99

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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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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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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Specific Double-Stranded RNA Interference in Undifferentiated Mouse Embryonic Stem Cells

Molecular and Cellular Biology ◽

10.1128/mcb.21.22.7807-7816.2001 ◽

2001 ◽

Vol 21 (22) ◽

pp. 7807-7816 ◽

Cited By ~ 154

Author(s):

Shicheng Yang ◽

Stephen Tutton ◽

Eric Pierce ◽

Kyonggeun Yoon

Keyword(s):

Gene Expression ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Es Cells ◽

Embryonic Stem ◽

Transient Transfection ◽

Interferon Response ◽

Green Fluorescent Protein Gene ◽

Double Stranded Rna ◽

Rnai Activity

ABSTRACT Specific mRNA degradation mediated by double-stranded RNA (dsRNA) interference (RNAi) is a powerful way of suppressing gene expression in plants, nematodes, and fungal, insect, and protozoan systems. However, only a few cases of RNAi have been reported in mammalian systems. Here, we investigated the feasibility of the RNAi strategy in several mammalian cells by using the enhanced green fluorescent protein gene as a target, either by in situ production of dsRNA from transient transfection of a plasmid harboring a 547-bp inverted repeat or by direct transfection of dsRNA made by in vitro transcription. Several mammalian cells including differentiated embryonic stem (ES) cells did not exhibit specific RNAi in transient transfection. This long dsRNA, however, was capable of inducing a sequence-specific RNAi for the episomal and chromosomal target gene in undifferentiated ES cells. dsRNA at 8.3 nM decreased the cognate gene expression up to 70%. However, RNAi activity was not permanent because it was more pronounced in early time points and diminished 5 days after transfection. Thus, undifferentiated ES cells may lack the interferon response, similar to mouse embryos and oocytes. Regardless of their apparent RNAi activity, however, cytoplasmic extracts from mammalian cells produced a small RNA of 21 to 22 nucleotides from the long dsRNA. Our results suggest that mammalian cells may possess RNAi activity but nonspecific activation of the interferon response by longer dsRNA may mask the specific RNAi. The findings offer an opportunity to use dsRNA for inhibition of gene expression in ES cells to study differentiation.

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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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