Investigation of the disposition of a markerchromosome in a mouse model

Acar-Perk, Bengi

Errors in the execution of meiosis can result in the generation of aneuploid gametes, which are associated with birth defects and infertility. According to published results; in 3% of chorionic villus sampling (CVS), the chromosomal abnormalities are being mosaics and do not mandatory affect the fetus (Astner et al., 1998) in human beings. So far, little is known about the molecular mechanisms of chromosome segregation and its leading to conditions. Therefore, we wanted to find out the distribution of aneuploidy (and translocation) in early embryogenesis in mouse trophoblast (TE) and inner cell mass (ICM) cells. Our main interest is, if aneuploidy are rather provable in the TE side and cause abortions, or in ICM side that results with defected babies or development in the early embryogenesis. In total, 241 female mice were sacrificed and 5 types of strains were examined in our experimental setting, which were B6D2F1, C3H/HEN, C57BL/6, BALB/c and CBA/CaHT(14;15)6Ca/J. The experimental group based on CBA/CaHT( 14;15)6Ca/J mouse strain, which has a balanced reciprocal translocation between chromosomes 14 and 15. Although there are no single chromosome 14 and 15, except the translocation chromosome, these mice were totally healthy and had breeding capacity. Due to the homozygosity, it is possible to observe the segregation of chromosomes. Trophectoderm (TE) biopsy, embryonic stem culture (ESC) and immunosurgery (IMS) protocols were evaluated for isolating individual ICM and TE cells. IMS and TE biopsy did not lead to our aim, because of their technical difficulties and false negative, positive results. Therefore, ESC technique was applied on 519 embryos and 462 (89%) embryos were developed healthy in culture. Specific FISH probe was developed for CBA/CaHT(14;15)6Ca/J translocated mice with four differently labelled BACs. We have demonstrated the feasibility of simultaneous fluorescence immunophenotyping and FISH to check the segregation of translocation and aneuploidy for the first time by isolating ICM and TE cells. FICTION (fluorescence immunophenotyping and interphase 168 cytogenetics as a tool for investigation of neoplasms) technique was successfully applied in 510 ICM and 510 TE cells of 73 mice embryos. Thus, we were able to visualize the antigen expression of Oct4 as ICM marker or Cdx as TE marker together with the multicolour FISH probe. Eighty % TE cells gave positive signals with Cdx labelling and 99% ICM cells gave positive signals with Oct4 labelling. The results from FICTION reveal that, all the cells (ICM+TE) of heterozygote translocated embryos carried the T(14;15)6Ca reciprocal translocation in a regular way. In heterozygote ICM cells we observed high numbers of aneuploidy than in heterozygote TE cells. Aneuploidies were observed by nondisjunction and unbalanced predivision. The level of mosaicism in the trophectoderm is lower than that seen in the inner cell mass of the blastocyst. This finding would pioneer the further investigations of cell repair or elimination. These results confirm those of Verlinsky et al. (2001). This, however, raises discussions about possible repair mechanisms once more. In investigating the marker chromosome in more detail, we could identify the break point in 14qE3 on a limited extension of 200 kb. Comparisons with available gene bank data reveal that up to now within this area no genes were annotated. In 15qA1 we could reduce the extension of breakpoint region on about 27 kb in the proteolytic enzyme gene ADAMTS12 (a disintegrin and metalloproteinase with thrombospondin motifs) playing a crucial rule in progression of vascularising as well as in cancer progression in human beings. With our results, this mouse strain lacking of ADAMTS12, will provide an essential tool to understand better the relevance of this enzyme in tumourassociated processes, asthma, arthritis, organogenesis, angiogenesis and reproduction.

Fehler in der Ausführung der Meiose können zur Herstellung aneuploider Gameten führen, die mit Geburtsdeffekten und Infertilität assoziieren. Laut veröffentlichter Ergebnisse, liegen in 3% der Proben von Chorionzotten Biopsien (CVS) chromosomale Abnormalitäten als Mosaik vor und beeinflussen nicht den menschlichen Fetus. Es ist bekannt, dass Fehler in der Chromosomenverteilung der Meiose zu Fehlbildungen und Infertilität führen, obwohl nur wenig über die molekularen Mechanismen der Chromosomensegregation und ihrer steuernden Bedingungen bekannt ist. Daher untersuchten wir die Verteilung von Aneuploidien (und Translokationen) in der frühen Mäuse-Embriogenese in Trophoblasten (TE) und der inneren Zellmasse (ICM). Unser Hauptinteresse galt der Frage, ob Aneuploidien eher im TE nachweisbar sind und Aborte verursachen oder eher in der ICM vorkommen und zu Fehlbildungen der Embryos führen. Insgesamt wurden 241 weibliche Mäuse der fünf unterschiedlichen Stämme B6D2F1, C3H/HEN, C57BL/6, BALB/c and CBA/CaHT(14;15)6Ca/J für diese Untersuchung verwandt. Die experimentelle Gruppe basiert auf dem CBA/CaHT( 14;15)6Ca/J Maus-Stamm, welcher eine homozygote, balancierte Translokation der Chromosomen 14 und 15 trägt. Die Mäuse sind völlig gesund und vermehren sich. Ihre Zellen tragen balancierte, reziproke Translokationschromosomen, die die Beobachtung ihrer Segregation ermöglichen. Biopsie Trophectoderm (TE), embryonische Stammzellkultur (ESC) und Immunosurgery (IMS) Protokolle wurden bezüglich ihrer Fähigkeit individuelle ICM und TE Zellen zu isolieren beurteilt. IMS und TE Biopsie führten wegen ihrer technischen Schwierigkeiten und falsch negativer, sowie falsch positiver Ergebnisse nicht zu unserem Ziel. Deshalb wurde die ESC-Technik bei 519 Embryos angewandt und 462 (89 %) der Embryos entwickelten sich gesund in Kultur.

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Acar-Perk, Bengi: Investigation of the disposition of a markerchromosome in a mouse model. 2009.

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