Identification of chromosomal-related abnormalities in Pfeiffer cell line using Fluorescence in situ hybridization

Studies that rely on fluorescence in situ hybridization (FISH) technique are of high quality and dependable results due to the fact that this technique is of high sensitivity, specificity, and speed which give FISH specific ability for detection of chromosomal abnormalities. FISH can be useful for detection of aneuploidy, characteristic gene fusions and loss of a chromosomal region. FISH can also be used as one of the beneficial tools in gene mapping and in detecting genetic abnormalities that might help in predicting cancers since they are considered to be precursors. The aim of this work was to inspect the presence of chromosomal abnormalities involving chromosomes 14 and 18 in the Pfeiffer cell line using FISH technology with specified whole chromosome paint of the above chromosomes. Key wards: FISH, bcl2 gene, chromosomal translocation INTRODUCTION Fluorescence in situ hybridization (FISH) was first introduced in 1969. Since that time FISH has been used for determination of viral DNA sequence, mRNA and chromosomal regions. Nowadays, FISH is one of the most powerful techniques available for both biologist and pathologist and it is widely used as routine technique in many laboratories. (1) FISH technology is based on the fact that single strand of DNA and RNA which labelled with fluorescence dye (probe) will hybridize to target molecular acids in neoplastic cells or tissues previously fixed on slides to form hybrids. In FISH techniques, the probe either labelled directly with the flourochrome such as Texas Red, FITC, Rhodamine, Cy2, Cy3, Cy5 and AMCA that are considered to be the most common. Or indirectly with a low molecular weight compound (hapten), and then subsequent amplification via a flourochrome conjugated antibody will be required including biotin that can be detected by streptavidin and digoxigenin that can be detected by specific antibodies. The direct technique is not as sensitive as the indirect one but it is quick and can be obtained with low background signal (1). In addition, the unique sequence probes, the whole chromosome paints, and the repetitive sequence probes are commonly used in FISH with specific applications for metaphase or interphase cells (1). Although this technique has been widely used since more than 30 years ago, the design and use of FISH probes remain one of the highly desired techniques. New application were recently added to the procedure including the use of tyramides and the rolling circle amplifications to be more advantageous in detecting small DNA target sequences of less than 1-3kb. Types of FISH technique includes fiber-FISH that is used to obtain high resolution mapping and determining the genomic rearrangements (2). Other types are known as Sky and M-FISH are useful in mapping of chromosomal breakpoints, unbalanced translocations awareness, marker chromosomes recognition in solid tumours and complex rearrangements characterization (3). Comparative genome organization (CGO) is a molecular cytogenetic method for the detection of chromosomal imbalances (4), and the procedure is mentioned below as seen in schematic (1). Schematic (1): Comparative genomic hybridization. MATERIALS AND METHODS: Pfeiffer cell line: Those cells were cultured after being obtained from lymphoma cells that belonged to diffuse large cell lymphoma patients during the leukemic phase (5). This disease is characterized by the presence of several chromosomal abnormalities that have been studied in details previously. Probes A whole chromosome paint specific for chromosome 14 labelled with biotin was used. Since biotin has no colour, streptavidin conjugated with cy3 was added. In addition to whole chromosome paint that as specific for chromosome 18 was directly labelled with fluorescein isothiocyanate (FITC) which gives green colour. Then, the probe was denatured at 65C° for 10 min on a heat block and incubated at 37 C° for 10min. Chromosome and nuclei preparations Five ml of the cell culture was centrifuged at 1200 rpm for 5 minutes, and then the supernatant was discarded. The pellet then was re-suspended by vortexing. After that, 5ml of hypotonic solution (KCL 0.075) was added to obtain in wards movement of water across cell membrane. As a result the cells got swelled and chromosomes in the metaphase got separated for harvesting purpose. Zainab Saad Ghafil Al-Raheem /J. Pharm. Sci. & Res. Vol. 11(3), 2019, 774-777