COBRA: combined binary ratio labeling of nucleic-acid probes for multi-color fluorescence in situ hybridization karyotyping

Szuhai, Károly; Tanke, Hans J.

doi:10.1038/nprot.2006.41

Cited by 72 publications

(67 citation statements)

References 21 publications

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“…The detailed protocol is described in Ref. 27. Briefly, probe sets were prepared by labeling of degenerate oligonucleotide-primed PCR-amplified individual chromosomes, isolated by flow sorting, with a specific combination of 1-4 fluorescent dyes (diethyl aminomethyl coumarin, Cy3, Cy5, and rhodamine green) (27).…”

Section: Methodsmentioning

confidence: 99%

Generation and Characterization of Transgenic Mice with the Full-length Human DMD Gene

Hoen

Meijer

Boer

et al. 2008

Journal of Biological Chemistry

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We report the generation of mice with an intact and functional copy of the 2.3-megabase human dystrophin gene (hDMD), the largest functional stretch of human DNA thus far integrated into a mouse chromosome. Yeast spheroplasts containing an artificial chromosome with the full-length hDMD gene were fused with mouse embryonic stem cells and were subsequently injected into mouse blastocysts to produce transgenic hDMD mice. Human-specific PCR, Southern blotting, and fluorescent in situ hybridization techniques demonstrated the intactness and stable chromosomal integration of the hDMD gene on mouse chromosome 5. Expression of the transgene was confirmed by RT-PCR and Western blotting. The tissue-specific expression pattern of the different DMD transcripts was maintained. However, the human Dp427p and Dp427m transcripts were expressed at 2-fold higher levels and human Dp427c and Dp260 transcripts were expressed at 2-and 4-fold lower levels than their endogenous counterparts. Ultimate functional proof of the hDMD transgene was obtained by crossing of hDMD mice with dystrophin-deficient mdx mice and dystrophin and utrophin-deficient mdx ؋ Utrn ؊/؊ mice. The hDMD transgene rescued the lethal dystrophic phenotype of the mdx ؋ Utrn ؊/؊ mice. All signs of muscular dystrophy disappeared in the rescued mice, as demonstrated by histological staining of muscle sections and gene expression profiling experiments. Currently, hDMD mice are extensively used for preclinical testing of sequence-specific therapeutics for the treatment of Duchenne muscular dystrophy. In addition, the hDMD mouse can be used to study the influence of the genomic context on deletion and recombination frequencies, genome stability, and gene expression regulation.

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Section: Methodsmentioning

confidence: 99%

Generation and Characterization of Transgenic Mice with the Full-length Human DMD Gene

Hoen

Meijer

Boer

et al. 2008

Journal of Biological Chemistry

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“…14 Hybridizations with individual BAC probes and whole chromosome painting (wcp) labeled with single fluorochromes were used to confirm the detected rearrangements. Chromosomal breakpoints were assigned by using inverted 4Ј,6-diamidino-2-phenylindole (DAPI; Downers Grove, IL) counterstained images together with the information derived from the short and long armspecific hybridizations.…”

Section: Combined Binary Ratio Fluorescence In Situ Hybridization (Comentioning

confidence: 99%

Heterogeneous and Complex Rearrangements of Chromosome Arm 6q in Chondromyxoid Fibroma

Romeo

Duim

Bridge

et al. 2010

The American Journal of Pathology

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Chondromyxoid fibroma (CMF) is an uncommon benign cartilaginous tumor of bone usually occurring during the second decade of life. CMF is associated with recurrent rearrangements of chromosome bands 6p23-25, 6q12-15, and 6q23-27. To delineate further the role and frequency of the involvement of three candidate regions (6q13, 6q23.3 and 6q24) in the pathogenesis of CMF, we studied a group of 43 cases using a molecular cytogenetic approach. Fluorescence in situ hybridization with probe sets bracketing the putative breakpoint regions was performed in 30 cases. The expression level of nearby candidate genes was studied by immunohistochemistry and quantitative RT-PCR in 24 and 23 cases, respectively. Whole-genome copy number screening was performed by array comparative genomic hybridization in 16 cases. Balanced and unbalanced rearrangements of 6q13 and 6q23.3 occurred in six and five cases, respectively, and a hemizygous deletion in 6q24 was found in five cases. Two known tumor suppressor genes map to the latter region: PLAGL1 and UTRN. However, neither of these two genes nor BCLAF1 and COL12A1, respectively located in 6q23.3 and 6q13, showed altered expression. Therefore, although rearrangements of chromosomal regions 6q13, 6q23.3, and 6q24 are common in CMF, the complexity of the changes precludes the use of a single fluorescence in situ hybridization probe set as an adjunct diagnostic tool. These data indicate that the genetic alterations in CMF are heterogeneous and are likely a result of a cryptic rearrangement beyond the resolution level of combined binary ratio fluorescence in situ hybridization or a point mutation.

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“…The 43-color FISH staining of every chromosome arm in a different color combination, digital imaging and analysis were performed as previously described (25).…”

Section: Combined Binary Ratio Fluorescence In Situ Hybridization (Comentioning

confidence: 99%

Establishment and detailed functional and molecular genetic characterisation of a novel sacral chordoma cell line, MUG-Chor1

Rinner

Froehlich²,

Buerger³

et al. 2011

Int J Oncol

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Abstract. Chordomas are rare, low to intermediate grade malignant bone tumors of the axial skeleton. Current treatment options are limited to surgical procedures, as chordomas are largely resistant to conventional radiation and chemotherapy. Cell lines are valuable tools for exploring molecular mechan isms involved in tumorigenesis and they have a fundamental impact on the development of new anticancer agents. To date, only two chordoma cell lines exist world-wide. In the present study we report a third chordoma cell line, MUG-Chor1, as well as corresponding cultured fibroblasts established from a recur rent morphologically 'classic' sacrococcygeal chordoma of a 58-year-old Caucasian female. The cells are brachyury-positive and have the characteristics of chordoma. The genetic profile of the primary chordoma and the established chordoma cell line was investigated during the culturing period (early and late passage). MUG-Chor1 is karyotypically, <2n>43-47,XX,del(3) (q1?) [11], +7,del(9)(p1?),der(9;15)(q10;q10),-10,+der(12)t(9;12) (p2?;q1?),der (12)t(12;19)(p;p)t(17;19)(q;q),-15,der(17;21) (q10;q10),der (20)t(10;20) (q25?26?;q11?12?),-21,-22[20]/ idemx2[5] and displays known, chordoma-typical genetic changes, such as chromosomal gains at T/brachyury locus (6q27), losses at 9p24.3-p13.1 (includes the CDKN2a/CDKN2b locus), 10p15.3-q23.32 (includes the PTEN locus) and losses of 10q25.2 (includes the PDCD4 locus). MUG-Chor1 bears a marked resemblance to chordomas in vivo and is, therefore, an optimal in vitro chordoma model.

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COBRA: combined binary ratio labeling of nucleic-acid probes for multi-color fluorescence in situ hybridization karyotyping

Cited by 72 publications

References 21 publications

Generation and Characterization of Transgenic Mice with the Full-length Human DMD Gene

Generation and Characterization of Transgenic Mice with the Full-length Human DMD Gene

Heterogeneous and Complex Rearrangements of Chromosome Arm 6q in Chondromyxoid Fibroma

Establishment and detailed functional and molecular genetic characterisation of a novel sacral chordoma cell line, MUG-Chor1

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