The Molecular Cytogenetic Core (MC) at the Albert Einstein College of Medicine provides tools for the preparation of human and murine samples suitable for molecular genetic and cytogenetic analysis of the entire genome. These tools include the establishment of EBV transformed cell lines; isolation of DNA and mRNA from a variety of tissue culture samples as well as primary biopsies; preparation of metaphase chromosomes suitable for fluorescence in situ hybridization (FISH) and Spectral Karyotyping (SKY) or whole chromosome paints for human and mouse genome. The core personnel is trained to hybridize commercial probes and to designed locus specific probes for regions of interest to investigators. All the probes are custom designed and in house generated.
- Metaphase preparation. We offer routine preparation of metaphase chromosomes by standard colcemid arrest and hypotonic treatment from human and mouse cell lines, peripheral blood and mouse spleen. High resolution chromosomes obtained by thymidine incorporation are also available.
- Standard karyotyping and chromosome counts. Chromosome counts for aneuploidy studies as well as full karyotyping based on inverted DAPI is provided for human and mouse.
- Generation of Probes and FISH hybridization. The MC offers services for both locus specific probes (BAC clones, plasmids > 5Kb) as well as chromosome painting probes (both human and mouse). We offer the possibility of selecting and ordering BAC clones for any genomic region based on the latest build of the genome. Probes can be generated in up to four colors (Spectrum Orange, Spectrum Aqua, Biotin- Cy5, Digoxigenin –Alexa 488 or Spectrum Green.
- Spectral Karyotyping (SKY). The MC is equipped to perform human and mouse SKY. SKY is a powerful 24-color, whole-chromosome genome wide painting assay that can detect chromosomal material of unknown origin, complex rearrangements, translocations, large deletions, duplications and aneuploidy.
- Epstein Barr Virus (EBV)-transformed human B-lymphoblastoid cell lines.
EBV infection in vitro causes transformation of B cells and generates B lymphoblastoid, permanent cell lines (LCLs). LCLs have been widely used for diagnosis of metabolic disorders. LCLs also have been used as models in various biological and medical studies as they can be easily established and continue proliferating for many generations to provide unlimited DNA/RNA. LCLs will be stored in liquid nitrogen. Our success rate is 90%.
- Genomic DNA isolation
The core is using Puregene Genomic DNA purification kit, Autogene 610L and traditional Phenol-Chloroform extraction methods to isolate DNA from whole blood, buffy coats, saliva, cell lines, buccal cells, tissues and serum. The 260/280 ratio is routinely of high quality of >1.7.
- Total RNA isolation
Qiagen RNeasy Mini Kit is used in the core for RNA isolation from whole blood, cell lines and tissues. We guarantee RNA samples of good quality and free of genomic DNA contamination or RNA degradation.
- Whole genome amplification
Whole genome amplification (WGA) uses DNA polymerase and random primers to amplify the entire genome. It can generate a large amount of DNA directly from a small sample. The core is using the REPLI-g whole genome amplification kit (Qiagen) for large scale and GenomiPhi DNA amplification Kit for small scale WGA.
- PBMC , serum isolation and storage
Density gradient centrifugation is used for PBMC isolation. The cell recovery viability is routinely 92%. PBMCs are stored in liquid nitrogen and serum will be kept at -80°C.
- Generation of hamster-human somatic cell hybrid cell lines
Somatic cell hybrids are formed by fusion of cells. Usually the fusion is achieved by polyethylene glycol (PEG) or a virus. After fusion, the new cell has temporarily a double chromosome complement that is gradually reduced by random expulsion of either hamster or human chromosomes. The remaining cells can be tested for the presence of a variety of gene products. The presence, or deficiency, of these products can be associated with the presence, or absence, of a particular chromosome, thus localizing function to site.
- Thawing and culture of frozen cell lines
The core provides different kinds of cells culture service for investigators.
- Cell repository
The facility has 8 large liquid nitrogen tanks for cell line storage and 2 Revcos for DNA storage.
- Shipment of cell lines and DNA
The core provides shipment service of cell lines and DNA.
A microscope for SKY images acquisition is located in the AIF (Analytical Imaging Facility, a shared resource at AECOM located in the Price Center room 210). The system include an Olympus BX51 with automatic stage, 6 positions fluorescent turret with specific Chroma filter sets, DIC and equipped with a Sensicam CCD cooled camera. The microscope is connected to a SpectraCube (Applied Spectral Imaging), this technology is based on spectral imaging, which combines two existing technologies, CCD-imaging and spectrometry. CCD imaging produces a finely detailed monochrome image of an object. Spectrometry, on the other hand, measures the spectrum of selected areas on the object and then displays each spectrum as a separate graph. The SpectraCube system is made up of an interferometer, a CCD-camera, a computer, and spectral image analysis software available to all GIF users. The SKY system is available to GIF users upon reservation and its use is charged by the AIF at standard fee. This system is connected to a DELL computer running the full ASI (http://www.spectral-imaging.com) acquisition and analysis package. This includes: FISH View for locus specific probes and chromosome painting analysis; CGH View for conventional CGH analysis; BandView for standard cytogenetic karyotyping of inverted DAPI metaphases; SKY View for SKY analysis.
The Olympus BX51 beside being connected to the SpectraCube is also and equipped with a Cooke SensicamQE camera with IPLab for image acquisition. Custom made script for multifocal image acquisition for the fluorophores of interest have been generate and tested for this system; thus for our purpose this function as a semi-automatic acquisition system.
The acquisition system has been recently fully upgraded with a SpotView C counting software for semi-automatic analysis of locus specific probes counting.
Metaphases for all experiments are prepared with the CDS-5 Cytogenetic Drying Chamber (Thermotron Industries). Locus Specific Probes are specifically labeled by nick translsation with modified dNTPs. Chromosome painting probes are generated by (Degenerate Oligonucleotide Primer) DOP-PCR using an Eppendorf Mastercycler ep.
|FISH hybridization (locus specific probes or chromosome painting) 1-3 color
|SKY (Human or Mouse)
|Selection of BAC clones and DNA isolation
|Karyotyping (human and mouse) 10 cells
|Probes (labeling only or chromosome paint for 5 reactions)
|EBV transformed human lymphocytes
|DNA isolation ( blood, SCOPE, spit, cultured cells, spit, cultured cells, frozen buffy coat, tissues)
|Sevarl manual and automatic protocols
|Automation DNA isolation from Hair
|Total RNA isolation (blood, cells, tissue...)
|QuickGene 810 RNA (blood, cells, tissue...)
|WGA (small scale and large scale)
|Cell culture T25-T75
|PBMC isolation 10/40 ml and 50/70 ml
|Eosinophil isolation (other blood cell subtypes, upon request)
|Cell Lines Liquid Nitrogen repository
|Picogreen DNA quantification
Price Center Room 407 and 413A, 1301 Morris Park Ave., Bronx, NY 10461 Please drop off your samples 413A
Cristina Montagna, PhD
Jidong Shan Ph.D.
Debbie Lewis B. S.
Yinghui Song B. S.
- Barzilai N, Atzmon G, Derby CA, Bauman JM, Lipton RB: A genotype of exceptional longevity is associated with preservation of cognitive function. Neurology 2006, 67(12):2170-2175.
- Jubinsky PT, Shanske AL, Pixley FJ, Montagna C, Short MK: A syndrome of holoprosencephaly, recurrent infections, and monocytosis. Am J Med Genet A 2006, 140(24):2742-2748.
- Babcock M, Yatsenko S, Hopkins J, Brenton M, Cao Q, de Jong P, Stankiewicz P, Lupski JR, Sikela JM, Morrow BE: Hominoid lineage specific amplification of low-copy repeats on 22q11.2 (LCR22s) associated with velo-cardio-facial/digeorge syndrome. Hum Mol Genet 2007, 16(21):2560-2571.
- Babcock M, Yatsenko S, Stankiewicz P, Lupski JR, Morrow BE: AT-rich repeats associated with chromosome 22q11.2 rearrangement disorders shape human genome architecture on Yq12. Genome Res 2007, 17(4):451-460.
- Samanich J, Lowes C, Burk R, Shanske S, Lu J, Shanske A, Morrow BE: Mutations in GJB2, GJB6, and mitochondrial DNA are rare in African American and Caribbean Hispanic individuals with hearing impairment. Am J Med Genet A 2007, 143A(8):830-838.
- Weaver BA, Silk AD, Montagna C, Verdier-Pinard P, Cleveland DW: Aneuploidy acts both oncogenically and as a tumor suppressor. Cancer Cell 2007, 11(1):25-36.
- Atzmon G, Pollin TI, Crandall J, Tanner K, Schechter CB, Scherer PE, Rincon M, Siegel G, Katz M, Lipton RB et al: Adiponectin levels and genotype: a potential regulator of life span in humans. J Gerontol A Biol Sci Med Sci 2008, 63(5):447-453.
- Battini L, Macip S, Fedorova E, Dikman S, Somlo S, Montagna C, Gusella GL: Loss of polycystin-1 causes centrosome amplification and genomic instability. Hum Mol Genet 2008, 17(18):2819-2833.
- Faggioli F, Sacco MG, Susani L, Montagna C, Vezzoni P: Cell fusion is a physiological process in mouse liver. Hepatology 2008, 48(5):1655-1664.
- Jhawer M, Goel S, Wilson AJ, Montagna C, Ling YH, Byun DS, Nasser S, Arango D, Shin J, Klampfer L et al: PIK3CA mutation/PTEN expression status predicts response of colon cancer cells to the epidermal growth factor receptor inhibitor cetuximab. Cancer Res 2008, 68(6):1953-1961.
- Roohi J, Cammer M, Montagna C, Hatchwell E: An improved method for generating BAC DNA suitable for FISH. Cytogenet Genome Res 2008, 121(1):7-9.
- Triplett AA, Montagna C, Wagner KU: A mammary-specific, long-range deletion on mouse chromosome 11 accelerates Brca1-associated mammary tumorigenesis. Neoplasia 2008, 10(12):1325-1334.
- Roohi J, Montagna C, Tegay DH, Palmer LE, DeVincent C, Pomeroy JC, Christian SL, Nowak N, Hatchwell E: Disruption of contactin 4 in three subjects with autism spectrum disorder. J Med Genet 2009, 46(3):176-182.
- Yuan Z, Shin J, Wilson A, Goel S, Ling YH, Ahmed N, Dopeso H, Jhawer M, Nasser S, Montagna C et al: An A13 repeat within the 3'-untranslated region of epidermal growth factor receptor (EGFR) is frequently mutated in microsatellite instability colon cancers and is associated with increased EGFR expression. Cancer Res 2009, 69(19):7811-7818.
- Shan J, Chobot-Rodd J, Castellanos R, Babcock M, Shanske A, Parikh SR, Morrow BE, Samanich J: GJB2 mutation spectrum in 209 hearing impaired individuals of predominantly Caribbean Hispanic and African descent. Int J Pediatr Otorhinolaryngol 2010, 74(6):611-618.