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Full Karyotype Interphase Cell Analysis.

ABSTRACT: Aneuploidy seems to play not only a decisive role in embryonal development but also in tumorigenesis where chromosomal and genomic instability reflect a universal feature of malignant tumors. The cost of whole genome sequencing has fallen significantly, but it is still prohibitive for many institutions and clinical settings. No applied, cost-effective, and efficient technique has been introduced yet aiming at research to assess the ploidy status of all 24 different human chromosomes in interphases simultaneously, especially in single cells. Here, we present the selection of human probe DNA and a technique using multistep fluorescence in situ hybridization (FISH) employing four sets of six labeled FISH probes able to delineate all 24 human chromosomes in interphase cells. This full karyotype analysis approach will provide additional diagnostic potential for single cell analysis. The use of spectral imaging (SIm) has enabled the use of up to eight different fluorochrome labels simultaneously. Thus, scoring can be easily assessed by visual inspection, because SIm permits computer-assigned and distinguishable pseudo-colors to each probe during image processing. This enables full karyotype analysis by FISH of single-cell interphase nuclei.

SUBMITTER: Baumgartner A

PROVIDER: S-EPMC6071177 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Full Karyotype Interphase Cell Analysis.

Baumgartner Adi A Ferlatte Hartshorne Christy C Polyzos Aris A AA Weier Heinz-Ulrich G HG Weier Jingly Fung JF O'Brien Ben B

The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 20180419 8

Aneuploidy seems to play not only a decisive role in embryonal development but also in tumorigenesis where chromosomal and genomic instability reflect a universal feature of malignant tumors. The cost of whole genome sequencing has fallen significantly, but it is still prohibitive for many institutions and clinical settings. No applied, cost-effective, and efficient technique has been introduced yet aiming at research to assess the ploidy status of all 24 different human chromosomes in interphas ...[more]

PMID: 29672206

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Project description:BackgroundKaryotype abnormalities are frequent in immortalized continuous cell lines either transformed or derived from primary tumors. Chromosomal rearrangements can cause dramatic changes in gene expression and affect cellular phenotype and behavior during in vitro culture. Structural variations of chromosomes in many continuous mammalian cell lines are well documented, but chromosome aberrations in cell lines from other vertebrate models often remain understudied. The chicken LSCC-HD3 cell line (HD3), generated from erythroid precursors, was used as an avian model for erythroid differentiation and lineage-specific gene expression. However, karyotype abnormalities in the HD3 cell line were not assessed. In the present study, we applied high-throughput chromosome conformation capture to analyze 3D genome organization and to detect chromosome rearrangements in the HD3 cell line.ResultsWe obtained Hi-C maps of genomic interactions for the HD3 cell line and compared A/B compartments and topologically associating domains between HD3 and several other cell types. By analysis of contact patterns in the Hi-C maps of HD3 cells, we identified more than 25 interchromosomal translocations of regions ≥ 200 kb on both micro- and macrochromosomes. We classified most of the observed translocations as unbalanced, leading to the formation of heteromorphic chromosomes. In many cases of microchromosome rearrangements, an entire microchromosome together with other macro- and microchromosomes participated in the emergence of a derivative chromosome, resembling "chromosomal fusions'' between acrocentric microchromosomes. Intrachromosomal inversions, deletions and duplications were also detected in HD3 cells. Several of the identified simple and complex chromosomal rearrangements, such as between GGA2 and GGA1qter; GGA5, GGA4p and GGA7p; GGA4q, GGA6 and GGA19; and duplication of the sex chromosome GGAW, were confirmed by FISH.ConclusionsIn the erythroid progenitor HD3 cell line, in contrast to mature and immature erythrocytes, the genome is organized into distinct topologically associating domains. The HD3 cell line has a severely rearranged karyotype with most of the chromosomes engaged in translocations and can be used in studies of genome structure-function relationships. Hi-C proved to be a reliable tool for simultaneous assessment of the spatial genome organization and chromosomal aberrations in karyotypes of birds with a large number of microchromosomes.

Project description:The karyotypes of Allium, a genus that comprises many crops and ornamental plants, are relatively poorly studied. To extend our knowledge on karyotype structure of the genus, the chromosomal organization of rRNA genes and CMA/DAPI bands was studied. Fluorescence in situ hybridization using 5S and 35S rDNA probes and banding methods (silver staining and CMA3/DAPI staining) were used to analyze the karyotypes of eight cultivated Allium L. species. Analyzed Allium taxa revealed three different basic chromosome numbers (x = 7, 8, 9) and three different ploidy levels (diploid, triploid, and tetraploid). The rDNA sites chromosomal organization is reported the first time for the six species (A. moly, A. oreophilum, A. karataviense, A. nigrum, A. sphaerocephalon, A. porrum). The Allium species that were analyzed showed a high level of interspecies polymorphism in the number and localization of the rDNA sites. The fluorescence in situ hybridization patterns of 35S rDNA sites were more polymorphic than those of the 5S rDNA in the diploid species. Several groups of similar chromosomes could be distinguished among the chromosomes that had rDNA sites in the polyploid species. Each of the groups had three chromosomes (triploid A. sphaerocephalon L.) or four chromosomes (tetraploid A. porrum L.) suggesting their autopolyploid origin. In the genomes of four of the analyzed species, only some of the 35S rDNA sites were transcriptionally active. Fluorochrome banding revealed that the CMA3+ bands were associated with the 35S rDNA sites in all of the species that were analyzed, except A. fistulosum L. in which positive CMA3+ bands were detected in the terminal position of all of the chromosome arms. The rDNA sequences, nucleolar organizer regions (NORs), and CMA/DAPI bands are very good chromosome markers that allowed to distinguished from two to five pairs of homologous chromosomes in analyzed Allium species. The karyotypes of the studied species could be clearly distinguished by the number and position of the rDNA sites, NORs, and CMA/DAPI bands, which revealed high interspecific differentiation among the taxa.

Project description:Background: Primary tumor Cell was an important tool for tumor research. Here, a new astrocytoma cell line SHG-140 was established and its proliferation, phenotype, karyotype, STR authentication, pathological characteristics, and characteristics of the cells' intrancranial xenografts of nude mice were studied. Methods: Primary SHG-140 culture was performed in DMEM/F12 medium with 10% FBS. Cell proliferation, karyotype analysis, cell immunofluorescence and STR authentication of SHG140 cells were performed. HE staining and immunohistochemistry, Whole oncogene high flux sequencing of the patient sample were carried out. SHG140 cells were injected into the brain of nude mice, HE staining and immunohistochemistry of intracranial xenograft tumor were detected. Results: Cell immunofluorescence demonstrated that SHG140 cells were positive for A2B5 (Glial precursors ganglioside), GFAP (Glial fibrillary acidic protein), Nestin, S-100 (Acid calcium bingding protein), Olig2 (Oligodendrocyte transcription factor 2) and Ki67 (Nuclear-associated antigen), cells negatively stained for Vimentin. Cell proliferation curve revealed that SHG140 proliferated slightly within 48 h, which then significantly proliferated to the fourth day. Karyotype analysis demonstrated its total number of chromosomes was 55, having trisomy of chromosome 6, 7, 8, 9 and X, and tetrad of chromosome 1 and 21, chromosomal deletion and rearrangement were observed. STR markers analysis showed the cells were derived from human male. SHG140 cells had tumorigenic properties - the intracranial injection of these cells into nude mice gave rise to growing tumors. We found that the glioma tissue was diffusively positive for GFAP, Nestin, slightly positive for Olig2, S-100; the positive rate of Ki-67 was 65% and negative for Vimentin. SHG140 cells were tumorigenic, GFAP, Nestin, S-100 Olig-2, the proliferation marker Ki-67 were expressed in its intracranial xenograft, Vimentin was negative expressed. Whole oncogene high flux sequencing of the patient tissue showed TP53, PTEN, IDH1 and PTCH1 mutation were existed. Conclusions: Our study showed that SHG140 was an astrocytoma glioma continuous cell line derived from a human adult male, having a strong tumorigenicity in nude mice, which made it wound be a useful model for the study of human glioblastoma multiforme.

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Full Karyotype Interphase Cell Analysis.

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Full Karyotype Interphase Cell Analysis.

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