Altering the Genome by Homologous Recombination

Capecchi, Mario R.

doi:10.1126/science.2660260

Cited by 1,904 publications

(969 citation statements)

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“…Culture conditions were subsequently established that allowed the isolation of pluripotent embryonic stem (ES) cells from mouse embryos [4]. The availability of mouse ES cells led to the development of gene targeting technology, which is widely used today [5]. Given the capacity of ES cells to generate virtually any cell type in the body, intense efforts have focused on harnessing the potential of human ES cells for medical applications.…”

Section: Introductionmentioning

confidence: 99%

Stem cell pluripotency and transcription factor Oct4

et al. 2002

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Mammalian cell totipotency is a subject that has fascinated scientists for generations. A long lasting question whether some of the somatic cells retains totipotency was answered by the cloning of Dolly at the end of the 20th century. The dawn of the 21 st has brought forward great expectations in harnessing the power of totipotentcy in medicine. Through stem cell biology, it is possible to generate any parts of the human body by stem cell engineering. Considerable resources will be devoted to harness the untapped potentials of stem cells in the foreseeable future which may transform medicine as we know today. At the molecular level, totipotency has been linked to a singular transcription factor and its expression appears to define whether a cell should be totipotent. Named Oct4, it can activate or repress the expression of various genes. Curiously, very little is known about Oct4 beyond its ability to regulate gene expression. The mechanism by which Oct4 specifies totipotency remains entirely unresolved. In this review, we summarize the structure and function of Oct4 and address issues related to Oct4 function in maintaining totipotency or pluripotency of embryonic stem cells.

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Section: Introductionmentioning

confidence: 99%

Stem cell pluripotency and transcription factor Oct4

et al. 2002

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“…The technique is highly successful when combined with a selection system in a cell system like embryonic stem cells, but is not successful for targeted therapeutic gene alteration in somatic cells. [1][2][3][4] Thus, alternative approaches have aimed to specifically target and alter chromosomal DNA with smaller structures. For targeting approaches without sequence constraint, short DNA fragments, 5 and modified or phosphothioate stabilized DNA oligonucleotides have been explored.…”

Section: Introductionmentioning

confidence: 99%

Branched oligonucleotides induce in vivo gene conversion of a mutated EGFP reporter

Olsen¹,

McKeen

Krauß³

2003

Gene Ther

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UKBranched oligonucleotides (b-oligonucleotides) based on a novel branching monomer were used for site-specific sequence alteration in vivo. With a stable integrated mutated enhanced green fluorescent protein (EGFP) template in Chinese hamster ovary cells, up to 0.1% EGFP-positive cells were counted after transfection with b-oligonucleotides. The presence of EGFP protein in converted cells was demonstrated by anti-EGFP immunocytochemistry. Genomic sequencing of converted cells showed in 40% of the analysed clones the corrected wild-type codon, while 9.3% of the sequences showed a corrected wild-type sequence and an additional collateral mutation. Despite the stable corrected genomic locus, converted cells entered selective apoptosis after 3-6 days. The cell line Irs-1 that is deficient in the homologous recombination pathway showed a reduced frequency of boligonucleotide-induced site-specific sequence conversion. The reduced conversion rates in the mutant cell line could be partly rescued by complementation with XRCC2 cDNA.

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“…Gene targeting by homologous recombination is a powerful method of specifically modifying a gene of interest used extensively for gene functional analysis in mice [1][2][3] . Gene targeting is accomplished in the mouse using embryonic stem (ES) cells, but in essentially all other species, ES cells suitable for gene targeting are not available.…”

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confidence: 99%

Sequential targeting of the genes encoding immunoglobulin-μ and prion protein in cattle

et al. 2004

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Gene targeting is accomplished using embryonic stem cells in the mouse but has been successful, only using primary somatic cells followed by embryonic cloning, in other species. Gene targeting in somatic cells versus embryonic stem cells is a challenge; consequently, there are few reported successes and none include the targeting of transcriptionally silent genes or double targeting to produce homozygotes. Here, we report a sequential gene targeting system for primary fibroblast cells that we used to knock out both alleles of a silent gene, the bovine gene encoding immunoglobulin-µ (IGHM), and produce both heterozygous and homozygous knockout calves. We also carried out sequential knockout targeting of both alleles of a gene that is active in fibroblasts, encoding the bovine prion protein (PRNP), in the same genetic line to produce doubly homozygous knockout fetuses. The sequential gene targeting system we used alleviates the need for germline transmission for complex genetic modifications and should be broadly applicable to gene functional analysis and to biomedical and agricultural applications.

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Altering the Genome by Homologous Recombination

Cited by 1,904 publications

References 32 publications

Stem cell pluripotency and transcription factor Oct4

Stem cell pluripotency and transcription factor Oct4

Branched oligonucleotides induce in vivo gene conversion of a mutated EGFP reporter

Sequential targeting of the genes encoding immunoglobulin-μ and prion protein in cattle

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