Gene targeting, principles, and practice in mammalian cells

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.

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.

Correction of β Thalassemia by Homologous Recombination in Embryonic Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 373-373 ◽  
Author(s):  
Thomas M. Ryan ◽  
Chiao-Wang Sun ◽  
Li-Chen Wu ◽  
Jin-Xiang Ren ◽  
Tim M. Townes
Keyword(s):  
Homologous Recombination ◽  
Es Cells ◽  
Globin Gene ◽  
Marker Gene ◽  
Embryonic Stem ◽  
Microcytic Anemia ◽  
Globin Genes ◽  
Wild Type ◽  
Distribution Width ◽  
Erythrocyte Morphology

Abstract Genetic correction of patient-derived embryonic stem (ES) cells is a powerful strategy for the treatment of hemoglobinopathies such as β thalassemia and sickle cell disease. One genetic strategy for the correction of β thalassemia is to replace mutant or deleted β-globin alleles with a wild-type gene by homologous recombination in ES cells. Thalassemic mice that mimic the disorder have been generated by targeted gene deletion of the adult murine β-globin genes (PNAS 92: 9259–9263). We derived ES cells from our β-globin knockout mice and produced genetically identical mutant mice by injecting the ES cells into tetraploid embryos. These cloned β thalassemic mice have a severe microcytic anemia characterized by a marked reduction of the erythrocyte mean corpuscular volume (MCV), hemoglobin level (Hb), and hematocrit (Hct), and a marked increase in reticulocytes and red cell distribution width (RDW) compared to cloned wild-type control animals. In contrast to the normochromic, normocytic erythrocytes of wild-type clones, erythrocytes in peripheral blood smears of β thalassemic mice were hypochromic and exhibit extreme anisopoikilocytosis. A targeting construct containing 8.7 kb of mouse homology flanking a human γ- and β-globin gene cassette and a hygromycin marker gene was electroporated into the β thalassemic ES cells. After selection, DNA from 48 ES cell colonies was analyzed by PCR to identify homologous recombinants. Nineteen colonies (40%) had correctly integrated the human globin genes into the deleted mouse β-globin locus. Correctly targeted cells were injected into tetraploid blastocysts to produce mice that are derived solely from the corrected ES cells. These cloned mice synthesize high levels of human β-globin polypeptide that corrects the α- to β-globin chain imbalance, thereby eliminating the thalassemic erythrocyte morphology. The MCV, Hb, Hct, RDW, and reticulocyte levels in the blood of these mice are normal. These results demonstrate that a severe hemoglobinopathy can be cured after targeted gene replacement of a mutant gene(s) with a wild-type allele by homologous recombination in ES cells.

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

Cytokine ◽  
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

scholarly journals Double-strand gap repair in a mammalian gene targeting reaction

1991 ◽  
Vol 11 (9) ◽  
pp. 4389-4397 ◽  
Author(s):  
V Valancius ◽  
O Smithies
Keyword(s):  
Stem Cells ◽  
Gene Targeting ◽  
Mammalian Cells ◽  
Embryonic Stem ◽  
Strand Break ◽  
Recombination Reaction ◽  
Hprt Gene ◽  
Chromosomal Locus ◽  
Mammalian Gene ◽  
Type Gene

To better understand the mechanism of homologous recombination in mammalian cells that facilitates gene targeting, we have analyzed the recombination reaction that inserts a plasmid into a homologous chromosomal locus in mouse embryonic stem cells. A partially deleted HPRT gene was targeted with various plasmids capable of correcting the mutation at this locus, and HPRT+ recombinants were directly selected in HAT medium. The structures of the recombinant loci were then determined by genomic Southern blot hybridizations. We demonstrate that plasmid gaps of 200, 600, and 2,500 bp are efficiently repaired during the integrative recombination reaction. Targeting plasmids that carry a double-strand break or gap in the region of DNA homologous to the target locus produce 33- to 140-fold more hypoxanthine-aminopterin-thymidine-resistant recombinants than did these same plasmids introduced in their uncut (supercoiled) forms. Our data suggest that double-strand gaps and breaks may be enlarged prior to the repair reaction since sequence heterologies carried by the incoming plasmids located close to them are often lost. These results extend the known similarities between mammalian and yeast recombination mechanisms and suggest several features of the insertional (O-type) gene targeting reaction that should be considered when one is designing mammalian gene targeting experiments.

scholarly journals Double-strand gap repair in a mammalian gene targeting reaction.

1991 ◽  
Vol 11 (9) ◽  
pp. 4389-4397 ◽  
Author(s):  
V Valancius ◽  
O Smithies
Keyword(s):  
Stem Cells ◽  
Gene Targeting ◽  
Mammalian Cells ◽  
Embryonic Stem ◽  
Strand Break ◽  
Recombination Reaction ◽  
Hprt Gene ◽  
Chromosomal Locus ◽  
Mammalian Gene ◽  
Type Gene

To better understand the mechanism of homologous recombination in mammalian cells that facilitates gene targeting, we have analyzed the recombination reaction that inserts a plasmid into a homologous chromosomal locus in mouse embryonic stem cells. A partially deleted HPRT gene was targeted with various plasmids capable of correcting the mutation at this locus, and HPRT+ recombinants were directly selected in HAT medium. The structures of the recombinant loci were then determined by genomic Southern blot hybridizations. We demonstrate that plasmid gaps of 200, 600, and 2,500 bp are efficiently repaired during the integrative recombination reaction. Targeting plasmids that carry a double-strand break or gap in the region of DNA homologous to the target locus produce 33- to 140-fold more hypoxanthine-aminopterin-thymidine-resistant recombinants than did these same plasmids introduced in their uncut (supercoiled) forms. Our data suggest that double-strand gaps and breaks may be enlarged prior to the repair reaction since sequence heterologies carried by the incoming plasmids located close to them are often lost. These results extend the known similarities between mammalian and yeast recombination mechanisms and suggest several features of the insertional (O-type) gene targeting reaction that should be considered when one is designing mammalian gene targeting experiments.

scholarly journals Cotransformation and gene targeting in mouse embryonic stem cells

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.

Production of targeted embryonic stem cell clones

1999 ◽  
Author(s):  
Michael P. Matise ◽  
Alexandra L. Joyner
Keyword(s):  
Cell Culture ◽  
Tissue Culture ◽  
Homologous Recombination ◽  
Gene Targeting ◽  
Es Cells ◽  
Embryonic Stem ◽  
Genetic Alterations ◽  
Functional Domains

The discovery that cloned DNA introduced into tissue culture cells can undergo homologous recombination at specific chromosomal loci has revolutionized our ability to study gene function in cell culture and in vivo. In theory, this technique, termed gene targeting, allows one to generate any type of mutation in any cloned gene. The kinds of mutations that can be created include null mutations, point mutations, deletions of specific functional domains, exchanges of functional domains from related genes, and gain-of-function mutations in which exogenous cDNA sequences are inserted adjacent to endogenous regulatory sequences. In principle, such specific genetic alterations can be made in any cell line growing in culture. However, not all cell types can be maintained in culture under the conditions necessary for transfection and selection. Over ten years ago, pluripotent embryonic stem (ES) cells derived from the inner cell mass (ICM) of mouse blastocyst stage embryos were isolated and conditions defined for their propogation and maintenance in culture (1, 2). ES cells resemble ICM cells in many respects, including their ability to contribute to all embryonic tissues in chimeric mice. Using stringent culture conditions, the embryonic developmental potential of ES cells can be maintained following genetic manipulations and after many passages in vitro. Furthermore, permanent mouse lines carrying genetic alterations introduced into ES cells can be obtained by transmitting the mutation through the germline by generating ES cell chimeras (described in Chapters 4 and 5). Thus, applying gene targeting technology to ES cells in culture affords researchers the opportunity to modify endogenous genes and study their function in vivo. In initial studies, one of the main challenges of gene targeting was to distinguish the rare homologous recombination events from more commonly occurring random integrations (discussed in Chapter 1). However, advances in cell culture and in selection schemes, in vector construction using isogenic DNA, and in the application of rapid screening procedures have made it possible to identify homologous recombination events efficiently. Since there are numerous publications available that describe basic tissue culture techniques in this chapter we will only describe techniques specific for ES cells.

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 Cotransformation and gene targeting in mouse embryonic stem cells.

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.

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.

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