Deletion and replacement of the mouse adult beta-globin genes by a "plug and socket" repeated targeting strategy.

J, Detloff, P; Lewis, Jada; W, John, S; Shehee, W R; Langenbach, R; Maeda, N; Smithies, Oliver

doi:10.1128/mcb.14.10.6936

Cited by 49 publications

(35 citation statements)

References 16 publications

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“…Several methods for the introduction of mutations in the mouse genome have been described (3,19,23,25,55,58,59). Despite the potential advantages, relatively few studies have used these approaches to determine the role of cis-acting sequences (7,20,37,50,57).…”

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confidence: 99%

A Gene-Targeting Approach Identifies a Function for the First Intron in Expression of the α1(I) Collagen Gene

Hormuzdi

Penttinen

Jaenisch

et al. 1998

Molecular and Cellular Biology

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The role of the first intron of the Col1A1 gene in the regulation of type I collagen synthesis remains uncertain and controversial despite numerous studies that have made use of transgenic and transfection experiments. To examine the importance of the first intron in regulation of the gene, we have used the double-replacement method of gene targeting to introduce, by homologous recombination in embryonic stem (ES) cells, a mutated Col1A1 allele (Col-Int⌬). The Col-Int⌬ allele contains a 1.3-kb deletion within intron I and is also marked by the introduction of a silent mutation that created an XhoI restriction site in exon 7. Targeted mice were generated from two independently derived ES cell clones. Mice carrying two copies of the mutated gene were born in the expected Mendelian ratio, developed normally, and showed no apparent abnormalities. We used heterozygous mice to determine whether expression of the mutated allele differs from that of the normal allele. For this purpose, we developed a reverse transcription-PCR assay which takes advantage of the XhoI polymorphism in exon 7. Our results indicate that in the skin, and in cultured cells derived from the skin, the intron plays little or no role in constitutive expression of collagen I. However, in the lungs of young mice, the mutated allele was expressed at about 75% of the level of the normal allele, and in the adult lung expression was decreased to less than 50%. These results were confirmed by RNase protection assays which demonstrated a two-to threefold decrease in Col1A1 mRNA in lungs of homozygous mutant mice. Surprisingly, in cultured cells derived from the lung, the mutated allele was expressed at a level similar to that of the wild-type allele. Our results also indicated an age-dependent requirement for the intact intron in expression of the Col1A1 gene in muscle. Since the intron is spliced normally, and since the mutant allele is expressed as well as the wild-type allele in the skin, reduced mRNA stability is unlikely to contribute to the reduction in transcript levels. We conclude that the first intron of the Col1A1 gene plays a tissue-specific and developmentally regulated role in transcriptional regulation of the gene. Our experiments demonstrate the utility of gene-targeting techniques that produce subtle mutations for studies of cis-acting elements in gene regulation.

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confidence: 99%

A Gene-Targeting Approach Identifies a Function for the First Intron in Expression of the α1(I) Collagen Gene

Hormuzdi

Penttinen

Jaenisch

et al. 1998

Molecular and Cellular Biology

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“…1). To facilitate recovery of the targeted clones, we combined to this scheme the plug-and-socket strategy developed by Detloff et al (11), which is based on the restoration by homologous recombination of a functional Hygro r gene. Introduction of the I-SceI recognition site into the endogenous villin locus.…”

Section: Resultsmentioning

confidence: 99%

“…To alleviate the screening of recombined clones during the second step, we used a strategy similar to the plug-and-socket strategy previously described by Detloff et al (11). They reported that using linearized constructs, insertion (O-type) targeting events occurred in their experiment, noticeably reducing the proportion of desired replacement recombinational (⍀-type) events among the drug-resistant clones.…”

Section: Discussionmentioning

confidence: 99%

I-SceI-Induced Gene Replacement at a Natural Locus in Embryonic Stem Cells

Cohen-Tannoudji

Robine

Choulika

et al. 1998

Molecular and Cellular Biology

117

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Gene targeting is a very powerful tool for studying mammalian development and physiology and for creating models of human diseases. In many instances, however, it is desirable to study different modifications of a target gene, but this is limited by the generally low frequency of homologous recombination in mammalian cells. We have developed a novel gene-targeting strategy in mouse embryonic stem cells that is based on the induction of endogenous gap repair processes at a defined location within the genome by induction of a double-strand break (DSB) in the gene to be mutated. This strategy was used to knock in an NH 2 -ezrin mutant in the villin gene, which encodes an actin-binding protein expressed in the brush border of the intestine and the kidney. To induce the DSB, an I-SceI yeast meganuclease restriction site was first introduced by gene targeting to the villin gene, followed by transient expression of I-SceI. The repair of the ensuing DSB was achieved with high efficiency (6 ؋ 10 ؊6 ) by a repair shuttle vector sharing only a 2.8-kb region of homology with the villin gene and no negative selection marker. Compared to conventional gene-targeting experiments at the villin locus, this represents a 100-fold stimulation of gene-targeting frequency, notwithstanding a much lower length of homology. This strategy will be very helpful in facilitating the targeted introduction of several types of mutations within a gene of interest.The ability to introduce specific alterations of endogenous genes into the germ line of mice via targeted mutagenesis in embryonic stem (ES) cells has represented a major breakthrough in mouse genetics. Gene inactivation has been widely used to examine the effects of loss of function in various biological processes such as development, cellular biology, and physiology. This has already permitted the accumulation of new insights into gene function and also the creation of mouse models of human genetic diseases. Introduction of subtle mutations at specific locations of the mammalian genome is also useful to refine genetic analysis and to produce models of genetic diseases which do not necessarily result from null mutations. Several strategies have been developed, each aimed at generating subtle mutations in a given gene (6). One common limitation to all current gene-targeting procedures is the low frequency of correct targeting. This becomes a serious problem especially with use of two successive rounds of targeting, a method common to several strategies used for the generation of mutated genes devoid of foreign selection sequences. Therefore, attempts have been made to increase the efficiency of gene targeting by several means, such as increasing the size of the region of homologies with the target locus, using isogenic genomic DNA, or improving the selection procedures (6).In this report, we present an alternative approach to overcome these limitations which relies on the observation that double-strand ends of broken chromosomes are highly recombinogenic (reviewed in reference 5). Double...

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“…The 'tag and exchange' strategy involves also two sequential gene targeting steps using two different replacement type vectors (Askew et al 1993). A modification of this protocol is the so-called 'plug and socket' approach developed by Detloff et al (1994). A much simpler method for introducing subtle mutations into the target gene has been developed by Reid et al (1991) and Davis et al (1992).…”

Section: Introduction Of Subtle Mutationsmentioning

confidence: 99%

Embryonic Stem Cells and Gene Targeting

Ledermann

2000

Experimental Physiology

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The development of gene targeting technology, the exchange of an endogenous allele of a target gene for a mutated copy via homologous recombination, and the application of this technique to murine embryonic stem cells has made it possible to alter the germ‐line of mice in a predetermined way. Gene targeting has enabled researchers to generate mouse strains with defined mutations in their genome allowing the analysis of gene function in vivo. This review presents the essential tools and methodologies used for gene targeting that have been developed over the past decade. Special emphasis has been laid on the available embryonic stem cell lines and the importance of the genetic background. Also, the state‐of‐the art of gene targeting approaches in species other than mice will be discussed.

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Deletion and replacement of the mouse adult beta-globin genes by a "plug and socket" repeated targeting strategy.

Cited by 49 publications

References 16 publications

A Gene-Targeting Approach Identifies a Function for the First Intron in Expression of the α1(I) Collagen Gene

A Gene-Targeting Approach Identifies a Function for the First Intron in Expression of the α1(I) Collagen Gene

I-SceI-Induced Gene Replacement at a Natural Locus in Embryonic Stem Cells

Embryonic Stem Cells and Gene Targeting

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