Gene Targeting to the Centromeric DNA of a Human Minichromosome

Raimondi, Elena; Balzaretti, Michela; Moralli, Daniela; Vagnarelli, Paola; Tredici, F.; Bensi, Mirella; Carli, L. De

doi:10.1089/hum.1996.7.9-1103

Cited by 24 publications

(23 citation statements)

References 25 publications

(15 reference statements)

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“…This can be explained by the presence of an active centromere. Several studies using linear human minichromosomes already showed that these segregated properly in human or hamster cells in the absence of selection (Farr et al 1995;Raimondi et al 1996;Harrington et al 1997;Ikeno et al 1998). There is some evidence, however, that the copy number of smaller minichromosomes (2.4 Mb) is more variable in human and hamster cell lines (Mills et al 1999).…”

Section: Discussion Mitotic Stability Of the Hcvmentioning

confidence: 99%

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Efficient Male and Female Germline Transmission of a Human Chromosomal Vector in Mice

Voet¹,

Vermeesch²,

Carens³

et al. 2001

Genome Res.

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A small accessory chromosome that was mitotically stable in human fibroblasts was transferred into the hprt − hamster cell line CH and developed as a human chromosomal vector (HCV) by the introduction of a selectable marker and the 3Ј end of an HPRT minigene preceded by a loxP sequence. This HCV is stably maintained in the hamster cell line. It consists mainly of alphoid sequences of human chromosome 20 and a fragment of human chromosome region 1p22, containing the tissue factor gene F3. The vector has an active centromere, and telomere sequences are lacking. By transfecting a plasmid containing the 5Ј end of HPRT and a Cre-encoding plasmid into the HCV + hamster cell line, the HPRT minigene was reconstituted by Cre-mediated recombination and expressed by the cells. The HCV was then transferred to male mouse R1-ES cells and it did segregate properly. Chimeras were generated containing the HCV as an independent chromosome in a proportion of the cells. Part of the male and female offspring of the chimeras did contain the HCV. The HCV + F1 animals harbored the extra chromosome in >80% of the cells. The HCV was present as an independent chromosome with an active centromere and the human F3 gene was expressed from the HCV in a human-tissue-specific manner. Both male and female F1 mice did transmit the HCV to F2 offspring as an independent chromosome with properties similar to the original vector. This modified small accessory chromosome, thus, shows the properties of a useful chromosomal vector: It segregates stably as an independent chromosome, sequences can be inserted in a controlled way and are expressed from the vector, and the HCV is transmitted through the male and female germline in mice.

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Section: Discussion Mitotic Stability Of the Hcvmentioning

confidence: 99%

“…Minichromosomes (all containing ␣ satellite repeats) of less than 2.5 Mb, thus, have been created. Finally, several authors explored the possibility of using naturally occurring minichromosomes (Raimondi et al 1996;Guiducci et al 1999).…”

mentioning

confidence: 99%

Efficient Male and Female Germline Transmission of a Human Chromosomal Vector in Mice

Voet¹,

Vermeesch²,

Carens³

et al. 2001

Genome Res.

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“…The labeled probes were resuspended in hybridization buffer (50% formamide, 10% Dextran sulfate, 1ϫ Denhardt's solution, 0.1% SDS, 40 mM Na 2 HPO 4 , pH 6.8, and 2ϫ SSC) at a final concentration of 5 g/ml and they were denatured at 70°C for 10 min. FISH on metaphase spreads was carried out essentially as previously reported (Raimondi et al, 1996). For FISH analysis on interphase heat-shocked HeLa cells, cells were rinsed with PBS and fixed in 4% formaldehyde for 15 min at room temperature.…”

Section: Fishmentioning

confidence: 99%

“…To confirm this conclusion, we took advantage of the fact that a human supernumerary mini-chromosome spanning the 9p13-q13 region had been previously characterized in our laboratory (Raimondi et al, 1991). MCH cells containing a single mini-chromosome as the only human chromosome (Raimondi et al, 1996) were therefore challenged for the ability to form stress bodies after heat shock. As hamsterϾhuman hybrid were heat shocked at 42°C for 1 h and after 1 h of recovery at 37°C, were fixed with formaldehyde, stained with the EDTA technique, and analyzed in electron microscopy.…”

Section: Stress-induced Snbs Colocalize With the Pericentromeric Hetementioning

confidence: 99%

Human Chromosomes 9, 12, and 15 Contain the Nucleation Sites of Stress-Induced Nuclear Bodies

Denegri

Moralli

Rocchi

et al. 2002

MBoC

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We previously reported the identification of a novel nuclear compartment detectable in heatshocked HeLa cells that we termed stress-induced Src-activated during mitosis nuclear body (SNB). This structure is the recruitment center for heat shock factor 1 and for a number of RNA processing factors, among a subset of Serine-Arginine splicing factors. In this article, we show that stress-induced SNBs are detectable in human but not in hamster cells. By means of hamsterϾhuman cell hybrids, we have identified three human chromosomes (9, 12, and 15) that are individually able to direct the formation of stress bodies in hamster cells. Similarly to stress-induced SNB, these bodies are sites of accumulation of hnRNP A1-interacting protein and heat shock factor 1, are usually associated to nucleoli, and consist of clusters of perichromatin granules. We show that the p13-q13 region of human chromosome 9 is sufficient to direct the formation of stress bodies in hamsterϾhuman cell hybrids. Fluorescence in situ hybridization experiments demonstrate that the pericentromeric heterochromatic q12 band of chromosome 9 and the centromeric regions of chromosomes 12 and 15 colocalize with stress-induced SNBs in human cells. Our data indicate that human chromosomes 9, 12, and 15 contain the nucleation sites of stress bodies in heat-shocked HeLa cells.

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“…The existence of spontaneously arising or radiation-induced human marker chromosomes offers an alternative starting point for deriving a chromosomebased vector system. [11][12][13][14][15][16][17] A further chromosome engineering technology under development relies upon integration of exogenous DNA into the centromere region of a host mouse or human chromosome. Amplification of DNA at the integration site can result in chromosome breakage events, producing new large chromosomes typically in the 60-400 Mb size range.…”

Section: Introductionmentioning

confidence: 99%