Patterns of integration of exogenous DNA sequences transfected into mammalian cells of primate and rodent origin

Colbère-Garapin, Florence; Ryhiner, Marie-Louise; Stephany, Isabelle; Kourilsky, Philippe; Garapin, Axel-Claude

doi:10.1016/0378-1119(86)90332-x

Cited by 28 publications

(11 citation statements)

References 19 publications

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“…A similar lack of success in deriving stably integrated YACs in human and mouse cell lines has been reported (28), although the reason for the failure in these cell lines is unclear. It has been proposed that the difficulties encountered (at least with human fibroblasts) are related to the limited efficiency of stable integration of human genomic DNA into the genome of human cells (29)(30)(31)(32)(33).…”

Section: Discussionmentioning

confidence: 99%

Substantial narrowing of the Niemann–Pick C candidate interval by yeast artificial chromosome complementation

Carstea²,

Cummings³

et al. 1997

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

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Niemann-Pick disease type C (NP-C) is an autosomal recessive lipidosis linked to chromosome 18q11-12, characterized by lysosomal accumulation of unesterified cholesterol and delayed induction of cholesterol-mediated homeostatic responses. This cellular phenotype is identifiable cytologically by filipin staining and biochemically by measurement of low-density lipoprotein-derived cholesterol esterification. The mutant Chinese hamster ovary cell line (CT60), which displays the NP-C cellular phenotype, was used as the recipient for a complementation assay after somatic cell fusions with normal and NP-C murine cells suggested that this Chinese hamster ovary cell line carries an alteration(s) in the hamster homolog(s) of NP-C. To narrow rapidly the candidate interval for NP-C, three overlapping yeast artificial chromosomes (YACs) spanning the 1 centimorgan human NP-C interval were introduced stably into CT60 cells and analyzed for correction of the cellular phenotype. Only YAC 911D5 complemented the NP-C phenotype, as evidenced by cytological and biochemical analyses, whereas no complementation was obtained from the other two YACs within the interval or from a YAC derived from chromosome 7. Fluorescent in situ hybridization indicated that YAC 911D5 was integrated at a single site per CT60 genome. These data substantially narrow the NP-C critical interval and should greatly simplify the identification of the gene responsible in mouse and man. This is the first demonstration of YAC complementation as a valuable adjunct strategy for positional cloning of a human gene.Niemann-Pick disease type C (NP-C) is an autosomal recessive neurodegenerative disorder with heterogeneous manifestations. The classic clinical profile includes variable hepatosplenomegaly, vertical supra-nuclear ophthalmoplegia, progressive ataxia, dystonia, and dementia (1). The metabolic lesion is characterized by lysosomal sequestration of low-density lipoprotein (LDL)-derived cholesterol leading to delayed cholesterol-mediated homeostatic responses (2, 3). Lysosomal cholesterol accumulation can be depicted cytochemically by the cholesterol-specific fluorescent dye, filipin. Normally, endocytosed LDL-derived cholesterol is mobilized from lysosomes to the endoplasmic reticulum for esterification. As a result, there is little free cholesterol accumulation in lysosomes detectable by filipin staining in normal cells. In contrast, in NP-C cells the lysosomal accumulation of the endocytosed LDL-derived free cholesterol results in a specific perinuclear filipin-staining pattern. Biochemically, the NP-C phenotype can most conveniently be monitored by LDL-induced cholesterol ester synthesis. Cholesterol ester synthesis is markedly stimulated by LDL in normal cells, but not in NP-C cells.

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Section: Discussionmentioning

confidence: 99%

Substantial narrowing of the Niemann–Pick C candidate interval by yeast artificial chromosome complementation

Carstea²,

Cummings³

et al. 1997

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

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“…In some cases, however, instability appears to be restricted to the time of integration. Many studies carried out in cultured cells 41,47,48,53 and transgenic mouse lines 43 have shown that while vectors can be physically rearranged initially, expansion of cell or mouse lines reveals no further rearrangements. This leads to the conclusion that the deletions and rearrangements observed are sometimes early events in the integration process.…”

Section: Recipient Genomic Loci Are Often Unstable After Illegitimatementioning

confidence: 99%

“…[49][50][51][52] Cell type differences in the amount of integrated DNA are reflected in experiments with human cells showing their genomes are 30-100 times less inclined to integrate as much exogenous DNA as rodent cells. [53][54][55][56] Finally, the transfection method can have an effect on the amount, and resulting form, of DNA integrating into a recipient genome. While calcium phosphate precipitation and microinjection result in relatively large amounts of DNA integrated in mouse cells, 39,41 electroporation shows a strong tendency for low copy numbers of integrated foreign DNA, and usually not in a standard head-to-tail array, but instead in a randomly arrayed structure.…”

Section: Introductionmentioning

confidence: 99%

Illegitimate DNA integration in mammalian cells

2003

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Foreign DNA integration is one of the most widely exploited cellular processes in molecular biology. Its technical use permits us to alter a cellular genome by incorporating a fragment of foreign DNA into the chromosomal DNA. This process employs the cell's own endogenous DNA modification and repair machinery. Two main classes of integration mechanisms exist: those that draw on sequence similarity between the foreign and genomic sequences to carry out homology-directed modifications, and the nonhomologous or 'illegitimate' insertion of foreign DNA into the genome. Gene therapy procedures can result in illegitimate integration of introduced sequences and thus pose a risk of unforeseeable genomic alterations. The choice of insertion site, the degree to which the foreign DNA and endogenous locus are modified before or during integration, and the resulting impact on structure, expression, and stability of the genome are all factors of illegitimate DNA integration that must be considered, in particular when designing genetic therapies.

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“…large amounts of tandemly arranged DNA (35,38) and has been reported to be accompanied by large rearrangements in the surrounding cell DNA (19,38). However, the amount of DNA integrated into human cells is generally much less (9,17,22,23,29), and integration commonly occurs without major rearrangements in the cell DNA (33). Analysis of the recombination events involved in integration in human cells has shown that the transfected sequences are inserted into small gaps (33), most likely by end ligation, a mechanism that is common in mammalian cells (39).…”

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confidence: 99%

Acquisition of telomere repeat sequences by transfected DNA integrated at the site of a chromosome break.

Murnane

1993

Mol. Cell. Biol.

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Previous analysis of plasmid DNA transfected into 108 cell clones demonstrated extensive polymorphism near the integration site in one clone. This polymorphism was apparent by Southern blot analysis as diffuse bands that extended over 30 kb. In the present study, nucleotide sequence analysis of cloned DNA from the integration site revealed telomere repeat sequences at the ends of the integrated plasmid DNA. The telomere repeat sequences at one end were located at the junction between the plasmid and cell DNA. The telomere repeat sequences at the other end were located in the opposite orientation in the polymorphic region and were shown by digestion with BAL 31 to be at the end of the chromosome. Telomere repeat sequences were not found at this location in the plasmid or parent cell DNA. Although the repeat sequences may have been acquired by recombination, a more likely explanation is that they were added to the ends of the plasmid by telomerase before integration. Comparison of the cell DNA before and after integration revealed that a chromosome break had occurred at the integration site, which was shown by fluorescent in situ hybridization to be located near the telomere of chromosome 13. These results demonstrate that chromosome breakage and rearrangement can result in interstitial telomere repeat sequences within the human genome. These sequences could promote genomic instability, because short repeat sequences can be recombinational hotspots. The results also show that

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Patterns of integration of exogenous DNA sequences transfected into mammalian cells of primate and rodent origin

Cited by 28 publications

References 19 publications

Substantial narrowing of the Niemann–Pick C candidate interval by yeast artificial chromosome complementation

Substantial narrowing of the Niemann–Pick C candidate interval by yeast artificial chromosome complementation

Illegitimate DNA integration in mammalian cells

Acquisition of telomere repeat sequences by transfected DNA integrated at the site of a chromosome break.

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