Reversal of Fortune for
            <i>Drosophila</i>
            Geneticists?

Engels, William R.

doi:10.1126/science.288.5473.1973

Cited by 21 publications

(5 citation statements)

References 13 publications

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“…First (Engels 2000), can genes that are not close to telomeres be targeted? Second (Anonymous 2000), can general methods be developed for targeted mutagenesis?…”

Section: Discussionmentioning

confidence: 99%

Targeted mutagenesis by homologous recombination inD. melanogaster

Rong¹,

Titen²,

Xie³

et al. 2002

Genes Dev.

307

345

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We used a recently developed method to produce mutant alleles of five endogenous Drosophila genes, including the homolog of the p53 tumor suppressor. Transgenic expression of the FLP site-specific recombinase and the I-SceI endonuclease generates extrachromosomal linear DNA molecules in vivo. These molecules undergo homologous recombination with the corresponding chromosomal locus to generate targeted alterations of the host genome. The results address several questions about the general utility of this technique. We show that genes not near telomeres can be efficiently targeted; that no knowledge of the mutant phenotype is needed for targeting; and that insertional mutations and allelic substitutions can be easily produced. We recently described a method for targeted modification of the Drosophila genome through homologous recombination (HR). The ability to engineer specific changes into the genome is a highly useful adjunct to genetic investigation in any organism, but especially in a species with a completely determined genome sequence such as Drosophila melanogaster (Adams et al. 2000). This procedure had, until recently, been lacking in Drosophila. In our previous reports, we targeted two genes, rescuing a mutant allele of the first and generating a mutant allele of the second (Rong and Golic 2000, 2001). At this time there is a clear need for demonstrations of the generality of this technique. That is, can a variety of genes in different locations be modified by HR? There is also a need for the development of techniques that can produce mutant alleles of target genes. In this work, we address both issues by applying new methods for targeted mutagenesis of five autosomal genes.A variety of schemes has been produced for targeted gene modification in organisms such as yeast and mice (Rothstein 1991;Muller 1999). However, these methods rely critically on the ability to culture single cells and carry out selections for rare events. Because the targeting technique we use occurs in whole animals, we devised variant approaches for introducing mutations into chromosomal genes. The methods are mechanistically similar to those developed for yeast and mouse, but procedurally quite different, as they do not rely on chemical selections. Instead, at each step, arbitrary genetic markers with simple visible phenotypes are used for genetic screening. In our previous experiments, the frequency of targeted gene modification through HR varied from ∼ 1 in 500 gametes to ∼ 1 in 30,000 gametes. These frequencies are easily within reach of the power provided by genetic screening.To perform gene targeting in flies we use transgenic expression of FLP site-specific recombinase and I-SceI endonuclease to generate a targeting donor molecule in vivo. This donor molecule is derived from a third transgenic element: a P element carrying DNA homologous to the target locus. Within the P element, FLP Recombinase Target sites (FRTs) flank a segment of DNA from the target locus, and an I-SceI recognition site is placed within the target-homolog...

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“…First (Engels 2000), can genes that are not close to telomeres be targeted? Second (Anonymous 2000), can general methods be developed for targeted mutagenesis?…”

Section: Discussionmentioning

confidence: 99%

Targeted mutagenesis by homologous recombination inD. melanogaster

Rong¹,

Titen²,

Xie³

et al. 2002

Genes Dev.

307

345

View full text Add to dashboard Cite

show abstract

“…BIR has not yet been studied at sites of Pelement excision or when site-specific endonucleases create DSBs, although this should be possible. Data from the Golic lab suggest that a few gene-targeting outcomes could involve BIR (Engels 2000;Rong and Golic 2000).…”

Section: Drosophilamentioning

confidence: 99%

Break-Induced DNA Replication

Anand

Lovett

Haber

2013

Cold Spring Harbor Perspectives in Biology

195

181

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“…Among other effects on the genome (such as genomic expansion due to repetitive DNA sequences, induction of hybrid dysgenesis, etc. ), this mobility can lead to mutation if the transposon lands in a functioning gene (insertional mutagenesis) 72–76. In some cases transposable elements have been shown to possess a reverse transcriptase‐like activity reminiscent of retroviruses; hence the designation retrotransposons 77–80.…”

Section: Clinical Implications Of Hypomethylationmentioning

confidence: 99%

Hypomethylation: One Side of a Larger Picture

Dunn

2003

Annals of the New York Academy of Sciences

146

100

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Hypomethylation signifies one end of a spectrum of DNA methylation states. In most cases hypomethylation refers to a relative state that represents a change from the "normal" methylation level. Hypomethylation, when approached from a topographical perspective, has been used to describe either overall decreases in the methylation status of the entire genome (global hypomethylation) or more localized relative demethylation of specific subsets of the genome, such as the promoter regions of protooncogenes or normally highly methylated repetitive sequences. Global hypomethylation accompanied by gene-specific hypermethylation is observed in at least two important settings: cancer and aging. Global hypomethylation is generally reflective of decreased methylation in CpGs dispersed throughout repetitive sequences as well as the bodies of genes. Hypomethylation of repetitive and parasitic DNA sequences correlates with a number of adverse outcomes. For example, decreased methylation of repetitive sequences in the satellite DNA of the pericentric region of chromosomes is associated with increased chromosomal rearrangements, a hallmark of cancer. Decreased methylation of proviral sequences can lead to reactivation and increased infectivity. However, hypomethylation in cancer can also affect the CpGs in the promoters of specific genes-namely, protooncogenes-leading to their overexpression and resulting in the functional outcome of increased cell proliferation. Thus, hypomethylation, in a variety of settings in which it represents a deviation from "normal," appears to correlate with progression to cancer and offers potential mechanisms to explain the carcinogenic process.

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Reversal of Fortune for Drosophila Geneticists?

Cited by 21 publications

References 13 publications

Targeted mutagenesis by homologous recombination inD. melanogaster

Targeted mutagenesis by homologous recombination inD. melanogaster

Break-Induced DNA Replication

Hypomethylation: One Side of a Larger Picture

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