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

2001 ◽  
Vol 73 (3) ◽  
pp. 365-383 ◽  
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.

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

1991 ◽  
Vol 11 (11) ◽  
pp. 5586-5591 ◽  
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.

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

1991 ◽  
Vol 11 (11) ◽  
pp. 5586-5591 ◽  
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.

Germ line transmission of an inactive N-myc allele generated by homologous recombination in mouse embryonic stem cells

1990 ◽  
Vol 10 (12) ◽  
pp. 6755-6758
Author(s):  
B R Stanton ◽  
S W Reid ◽  
L F Parada
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Homologous Recombination ◽  
Embryonic Development ◽  
Cell Lines ◽  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Germ Line Transmission

We have disrupted one allele of the N-myc locus in mouse embryonic stem (ES) cells by using homologous recombination techniques and have obtained germ line transmission of null N-myc ES cell lines with transmission of the null N-myc allele to the offspring. The creation of mice with a deficient N-myc allele will allow the generation of offspring bearing null N-myc alleles in both chromosomes and permit study of the role that this proto-oncogene plays in embryonic development.

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

1994 ◽  
Vol 14 (2) ◽  
pp. 1009-1016 ◽  
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.

scholarly journals Germ line transmission of an inactive N-myc allele generated by homologous recombination in mouse embryonic stem cells.

1990 ◽  
Vol 10 (12) ◽  
pp. 6755-6758 ◽  
Author(s):  
B R Stanton ◽  
S W Reid ◽  
L F Parada
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Homologous Recombination ◽  
Embryonic Development ◽  
Cell Lines ◽  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Germ Line Transmission

We have disrupted one allele of the N-myc locus in mouse embryonic stem (ES) cells by using homologous recombination techniques and have obtained germ line transmission of null N-myc ES cell lines with transmission of the null N-myc allele to the offspring. The creation of mice with a deficient N-myc allele will allow the generation of offspring bearing null N-myc alleles in both chromosomes and permit study of the role that this proto-oncogene plays in embryonic development.

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

1994 ◽  
Vol 14 (2) ◽  
pp. 1009-1016
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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