Insertion of DNA sequences into the human chromosomal β-globin locus by homologous recombination

Smithies, Oliver; Gregg, Ronald G.; Boggs, Sallie S.; Koralewski, Michael A.; Kucherlapati, Raju

doi:10.1038/317230a0

Cited by 946 publications

(446 citation statements)

References 24 publications

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“…Transgenic mice may be created 'classically' through (1) direct pro-nuclear injection of exogenous DNA into fertilized zygotes (Gordon and Ruddle, 1983;Brinster et al, 1985;Palmiter and Brinster, 1986), implantation into a pseudo-pregnant female-generating transgenic progeny or (2) via injection of genetically modified mouse embryonic stem (ES) cells into a blastocyst (Doetschman et al, 1985;Gossler et al, 1986;Robertson et al, 1986) (embryonic day 4.5) with (3) embryonic retroviral infection (Jaenisch, 1980;Jaenisch et al, 1981;Soriano and Jaenisch, 1986), representing a further alternative. While direct pronuclear injection of exogenous DNA results in indiscriminate integration into the genome and is reliant upon overexpression of the transgene to generate a phenotype, ES cells can be genetically tailored via homologous recombination (Smithies et al, 1985) (that is location and enzymatic recombination of an exogenous DNA fragment into the homologous endogenous sequence; Figure 1a and Supplementary Information) in vitro prior to blastocoel insertion, representing the superior technique. Following electroporation of exogenous DNA into the ES cell and culture, selected cells containing the targeted allele are subsequently injected into blastocysts and implanted into pseudo-pregnant mothers ( Figure 1b and Supplementary Information).…”

Section: Transgenic Micementioning

confidence: 99%

Review: genetic models of acute myeloid leukaemia

2008

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The use of genetically engineered mice (GEM) have been critical in understanding disease states such as cancer, and none more so than acute myelogenous leukaemia (AML), a disease characterized by over 100 distinct chromosomal translocations. A substantial proportion of cases exhibiting recurrent reciprocal translocations at diagnosis, such as t(8;21) or t(15;17) have been exhaustively studied and are currently employed in clinical diagnosis. However, a definitive conclusion regarding the leukaemogenic potential of defined transgenes for this disease remains elusive. While it is increasingly apparent that a number of cooperating mutations are necessary to develop a leukaemic phenotype, the number of models reflecting these synergisms remains few. Furthermore, little emphasis has been paid to the effect of chromosomal translocations other than recurrent genetic abnormalities, with no models reflecting the multiple abnormalities observed in high-risk cases of AML accounting for 8-10% of adult AML. Here we review the differing technologies employed in generation of GEM of AML. We discuss the relevance of GEM AML from embryonic stem cell-mediated (for example retinoic acid receptor-a fusions and AML1/ETO) models; through to the valuable retroviral-mediated gene transfer models. The latter have been used to great effect in defining the transforming properties of chromosomal translocation products such as MLL (found in 5-6% of all AML cases) and NUP98 (denoting poor prognosis in therapy-related disease) and particularly when co-transduced with bad prognostic factors such as Flt3 mutations. Finally, we comment on the emergence of newer transduction technologies, which can regulate the level of expression to defined cell lineages in both primary murine and human xenografts, and discuss how combining multiple genetic modalities, more relevant models of this complex disease are being generated.

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Section: Transgenic Micementioning

confidence: 99%

Review: genetic models of acute myeloid leukaemia

2008

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“…In yeast, DSB repair occurs largely by homologous recombination (4)(5)(6)(7). Although capable of homologous recombination, higher eukaryotic species repair DSBs primarily by a process of nonhomologous or illegitimate recombination (8)(9)(10)(11)(12)(13).…”

mentioning

confidence: 99%

DNA looping by Ku and the DNA-dependent protein kinase

Cary

Peterson

Wang

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

228

138

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The DNA-dependent protein kinase (DNA-PK) is required for DNA double-strand break (DSB) repair and immunoglobulin gene rearrangement and may play a role in the regulation of transcription. The DNA-PK holoenzyme is composed of three polypeptide subunits: the DNA binding Ku70͞86 heterodimer and an Ϸ460-kDa catalytic subunit (DNA-PKcs). DNA-PK has been hypothesized to assemble at DNA DSBs and play structural as well as signal transduction roles in DSB repair. Recent advances in atomic force microscopy (AFM) have resulted in a technology capable of producing high resolution images of native protein and proteinnucleic acid complexes without staining or metal coating. The AFM provides a rapid and direct means of probing the protein-nucleic acid interactions responsible for DNA repair and genetic regulation. Here we have employed AFM as well as electron microscopy to visualize Ku and DNA-PK in association with DNA. A significant number of DNA molecules formed loops in the presence of Ku. DNA looping appeared to be sequence-independent and unaffected by the presence of DNA-PKcs. Gel filtration of Ku in the absence and the presence of DNA indicates that Ku does not form nonspecific aggregates. We conclude that, when bound to DNA, Ku is capable of self-association. These findings suggest that Ku binding at DNA DSBs will result in Ku self-association and a physical tethering of the broken DNA strands.

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“…As mentioned, gene deletion by homologous recombination (HR) has been used in reverse genetics experiments for many years (Smithies et al 1985). In its most basic form it comprises a drug selection marker and a DNA sequence with homology to the locus of interest in the target cell (Fig.…”

Section: Therapuetic Protein Productionmentioning

confidence: 99%

“…Techniques such as gene deletion (knockout) by homologous recombination were developed over 20 years ago to study gene function in the mouse (Smithies et al 1985). In the research laboratory this method remains the definitive means of silencing genes (notwithstanding the widespread use of RNAi for knockdown) though many additional features can now be included in the common-or-garden knockout construct-these will be discussed in more detail later.…”

Section: Introductionmentioning

confidence: 99%

Targeted genetic modification of cell lines for recombinant protein production

2007

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Considerable increases in productivity have been achieved in biopharmaceutical production processes over the last two decades. Much of this has been a result of improvements in media formulation and process development. Though advances have been made in cell line development, there remains considerable opportunity for improvement in this area. The wealth of transcriptional and proteomic data being generated currently hold the promise of specific molecular interventions to improve the performance of production cell lines in the bioreactor. Achieving this-particularly for multi-gene modification-will require specific, targeted and controlled genetic manipulation of these cells. This review considers some of the current and potential future techniques that might be employed to realise this goal.

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Insertion of DNA sequences into the human chromosomal β-globin locus by homologous recombination

Cited by 946 publications

References 24 publications

Review: genetic models of acute myeloid leukaemia

Review: genetic models of acute myeloid leukaemia

DNA looping by Ku and the DNA-dependent protein kinase

Targeted genetic modification of cell lines for recombinant protein production

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