Investigation of the effects of different cryopreservation parameters on the genome of 51/4 hpf zebrafish (Danio rerio) embryos

2.50
Hdl Handle:
http://hdl.handle.net/10547/322165
Title:
Investigation of the effects of different cryopreservation parameters on the genome of 51/4 hpf zebrafish (Danio rerio) embryos
Authors:
Ahmed, Raju
Abstract:
In recent years, numerous studies have linked cryopreservation with increased occurrence of mutations, DNA fragmentation and the event of apoptosis in biological objects. However, the evidence emerged from such studies is somewhat inconclusive. The current study, therefore, aimed to analyse the DNA damage response (DDR) from the cryopreserved cells in order to characterise the nature of the putative DNA damage. The study set out to investigate the effects of different cryopreservation parameters on the genome in terms of double strand breaks (DSBs), single strand breaks (SSBs), and various forms of sequence alteration using 5¼ hour post fertilisation (hpf) zebrafish (Danio rerio) embryos. The experimental conditions under which the investigation was carried out were short term chilling at 0˚C, treatment with two cryoprotective agents (CPA), namely, MeOH and Me2SO, and cooling to -35˚C. Assays for detecting DSB-activated DDR proteins and SSB-activated DDR proteins in 5¼ hpf zebrafish (Danio rerio) were developed and then utilised to investigate the occurrence of DSBs and SSBs in the genome of the embryos treated with the experimental conditions. The study then analysed the expression profiles of a set of genes unique to the base excision repair (BER), nucleotide excision repair (NER) and mismatch repair (MMR) pathways as indicators of the occurrence of various forms of sequence alterations in the genome of the embryos treated with the experimental conditions. It was found that chilling and CPA treatment did not induce DSBs or SSBs but up-regulated the MMR and BER, respectively. CPA treatment also down-regulated the NER and the MMR mechanisms. Cooling, on the contrary, did not induce DSBs but induced SSBs in the genome, which were repaired when the embryos were provided with a recovery time. Cooling also up-regulated the NER and the BER mechanisms in the embryos. The overall finding of the study indicated that the experimental conditions increased the occurrence of various single stranded DNA lesions in the genome of the embryos. The present study provided important insights into how eukaryotic cells respond to different cryopreservation parameters, which will significantly enhance the current knowledge of the effects of cryopreservation on the genome of biological objects.
Affiliation:
University of Bedfordshire
Citation:
Ahmed, R. (2013) 'Investigation of the effects of different cryopreservation parameters on the genome of 51/4 hpf zebrafish (Danio rerio) embryos'. PhD thesis. University of Bedfordshire.
Publisher:
University of Bedfordshire
Issue Date:
Jun-2013
URI:
http://hdl.handle.net/10547/322165
Type:
Thesis or dissertation
Language:
en
Description:
A thesis is submitted to the University of Bedfordshire in accordance with the requirements for the degree of Doctor of Philosophy
Appears in Collections:
PhD e-theses

Full metadata record

DC FieldValue Language
dc.contributor.authorAhmed, Rajuen
dc.date.accessioned2014-06-23T10:43:11Z-
dc.date.available2014-06-23T10:43:11Z-
dc.date.issued2013-06-
dc.identifier.citationAhmed, R. (2013) 'Investigation of the effects of different cryopreservation parameters on the genome of 51/4 hpf zebrafish (Danio rerio) embryos'. PhD thesis. University of Bedfordshire.en
dc.identifier.urihttp://hdl.handle.net/10547/322165-
dc.descriptionA thesis is submitted to the University of Bedfordshire in accordance with the requirements for the degree of Doctor of Philosophyen
dc.description.abstractIn recent years, numerous studies have linked cryopreservation with increased occurrence of mutations, DNA fragmentation and the event of apoptosis in biological objects. However, the evidence emerged from such studies is somewhat inconclusive. The current study, therefore, aimed to analyse the DNA damage response (DDR) from the cryopreserved cells in order to characterise the nature of the putative DNA damage. The study set out to investigate the effects of different cryopreservation parameters on the genome in terms of double strand breaks (DSBs), single strand breaks (SSBs), and various forms of sequence alteration using 5¼ hour post fertilisation (hpf) zebrafish (Danio rerio) embryos. The experimental conditions under which the investigation was carried out were short term chilling at 0˚C, treatment with two cryoprotective agents (CPA), namely, MeOH and Me2SO, and cooling to -35˚C. Assays for detecting DSB-activated DDR proteins and SSB-activated DDR proteins in 5¼ hpf zebrafish (Danio rerio) were developed and then utilised to investigate the occurrence of DSBs and SSBs in the genome of the embryos treated with the experimental conditions. The study then analysed the expression profiles of a set of genes unique to the base excision repair (BER), nucleotide excision repair (NER) and mismatch repair (MMR) pathways as indicators of the occurrence of various forms of sequence alterations in the genome of the embryos treated with the experimental conditions. It was found that chilling and CPA treatment did not induce DSBs or SSBs but up-regulated the MMR and BER, respectively. CPA treatment also down-regulated the NER and the MMR mechanisms. Cooling, on the contrary, did not induce DSBs but induced SSBs in the genome, which were repaired when the embryos were provided with a recovery time. Cooling also up-regulated the NER and the BER mechanisms in the embryos. The overall finding of the study indicated that the experimental conditions increased the occurrence of various single stranded DNA lesions in the genome of the embryos. The present study provided important insights into how eukaryotic cells respond to different cryopreservation parameters, which will significantly enhance the current knowledge of the effects of cryopreservation on the genome of biological objects.en
dc.language.isoenen
dc.publisherUniversity of Bedfordshireen
dc.subjectC410 Applied Geneticsen
dc.subjectcryopreservationen
dc.subjectzebrafishen
dc.subjectDanio rerioen
dc.subjectembryosen
dc.titleInvestigation of the effects of different cryopreservation parameters on the genome of 51/4 hpf zebrafish (Danio rerio) embryosen
dc.typeThesis or dissertationen
dc.contributor.departmentUniversity of Bedfordshireen
dc.type.qualificationnamePhDen_GB
dc.type.qualificationlevelPhDen
dc.publisher.institutionUniversity of Bedfordshireen
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