TRISOMY 8 MOSAICISM: CELL CYCLE KINETICS AND ...

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(Swisshelm et al. 1981; Guichet et al. 1995; Miller et al. 1997; Jordan et al. 1998; Webb et al. 1998). Fluorescence In Situ Hybridization Fluorescence in situ hybridization (FISH) is a molecular technique first introduced in the late 1970’s (Trask 1991). It can be used for chromosomal analysis and identification and clinical diagnostic purposes. FISH is widely used due to several factors including: I.) DNA can be investigated in metaphase spreads and interphase nuclei II.) The technique is reliable and repeatable due to commercially available probes and fluorescent reagents III.) A broad range of DNA sizes can be analyzed including entire genome, chromosome, sub-regions, or single copy sequences (Trask 1991). FISH involves annealing of DNA probes to subject DNA. Probes are selected based on initial diagnosis or suspected chromosome abnormalities. Centromeric alpha-satellite probes can be used for metaphase and interphase analysis (Moore et al. 2000). These centromeric probes are chosen in cases where patients are suspected to have whole chromosomal abnormalities, since the probe will only determine if the chromosome is present and not identify specific chromosomal regions. Probes for chromosome X may also be used to rule out maternal contamination in placental tissue when the patient is male, on the basis of males resulting in one X signal and females with two X signals. Cell Cycle Kinetics Growth can be easily measured by incorporation of 5’-bromo-2’-deoxyuridine (BrdU) in tissue culture. BrdU is a base analog that is substituted for thymidine in the 6
DNA undergoing synthesis. Once a sample is subcultured, BrdU is added and will become incorporated into the genome of the replicating cells (Bonhoeffer 2000). This newly replicated cell will continue to undergo divisions until the cell culture growth is terminated. The BrdU is detected in the chromosomes by a fluorescent stain on metaphase spreads. Cell divisions are determined by the amount of BrdU incorporation among chromatids. Equal staining among chromatids represents one division (singly-substituted double stranded DNA). Two divisions are seen as half the chromatids stained pale (doubly substituted) and the other half stained dark. Three divisions are recognized by three-fourths of the chromatids stained pale and one-fourth stained dark. Cell cycle kinetics analysis allows for detection of growth rate differences between different tissues and in normal versus abnormal cells. Purpose The first purpose of the study is to determine the level of mosaicism among several embryonic and extra-embryonic tissues from a male patient. Once the percentages of normal and trisomy 8 cells are determined for each tissue statistical analysis will be performed. Since a selective growth advantage is known to occur for normal cells over trisomy 8 cells, the second purpose of the study is to investigate cell cycle kinetics for each tissue. Cell cycle kinetics will determine the growth rates of the tissues and statistical analysis performed to denote significant differences in division times between normal and trisomy 8 cells. 7
Page 1 and 2: TRISOMY 8 MOSAICISM: CELL CYCLE KIN
Page 3 and 4: TABLE OF CONTENTS iii Page Abstract
Page 5 and 6: Trisomy 8 INTRODUCTION Trisomy 8 (T
Page 7 and 8: proliferates and thus mosaicism res
Page 9: Prenatal Testing Prenatal testing i
Page 13 and 14: centrifugation, aspiration, and fix
Page 15 and 16: insed with water and allowed to air
Page 17 and 18: fixative, and the centrifugation an
Page 19 and 20: FISH slide preparation Day 1 Slides
Page 21 and 22: Case Report RESULTS Amniocentesis,
Page 23 and 24: Figure 2 Trisomy 8 [46, XY, +8] Kar
Page 25 and 26: When comparing embryonic tissues, c
Page 27 and 28: Figure 4 FISH Trisomy 8 [46, XY, +8
Page 29 and 30: There was a notable difference betw
Page 31 and 32: For each tissue the metaphase and i
Page 33 and 34: Figure 6 Second Division - Cell Cyc
Page 35 and 36: Table 4. Division Rates for Normal
Page 37 and 38: Origin of Trisomy 8 cells DISCUSSIO
Page 39 and 40: original finding of La Marche in th
Page 41 and 42: Pallister Killian patients with the
Page 43 and 44: BIBLIOGRAPHY Berry AC, Mutton DE, a
Page 45: Thornberg Reeser SL and Wenger SL.

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