PCNA (Proliferating Cell nuclear antigen) is a key protein in the regulation of many cellular processes, mainly linked to nucleic acid metabolism; it is considered a processivity factor for the DNA polymerases delta and epsilon, playing an important role in DNA replication; it is also involved in many other pathways, such as Okazaki fragment processing, DNA repair and translesion DNA synthesis, chromatin remodelling, DNA methylation and cell cycle regulation. Since PCNA does not possess any enzymatic activity, its functions are carried out through the interaction with many other proteins working in the different pathways mentioned before. Because of its presence in several intracellular mechanisms and because of its numerous protein interactions, PCNA is also involved in several serious human pathologies such as Systemic Lupus Erythematosus (SLE) and Cancer.The involvement of PCNA in such pathologies underlie the importance of performing further studies on this protein. Particularly important are the studies of PCNA gene mutations.The mouse transgenic technology, that allows the study of the phenotypic effect of gene modification in vivo, represents a great tool for the functional study of gene mutations. This technology can be used for the creation of knock-out mutants (with the aim to either delete part of the DNA sequence or insert irrelevant DNA sequence information to disrupt the expression of a specific genetic locus), or knock-in mutants (aimed to alter the genetic locus of interest via a substitution of DNA sequences or by the addition of a sequence that is not normally found on that genetic locus). One crucial step of this procedure is represented by the screening and the identification of recombinant ES cells. This screening must not only precise and specific, but also simple. The method of choice for the generation of the knock-in ES cells was the Cre-LoxP system and vector that was composed of a positive selection marker cassette (Neor) and a construct containing a point mutation in the Pcna coding sequence, all flanked by loxP sites. My thesis is about genomic analysis of mouse embryonic stem (ES) cell lines bearing Pcna mutations and my work wants to provide a valid and precise screening strategy that can be applied for the selection of the recombinant ES cells following the knock-in procedure. The strategy developed is based on two main methods: screening by PCR analysis and screening by Southern blot analysis. The transgenic mouse created using this modified ES cells will allow the analysis of the mutated Pcna gene, providing a new model for study the impact of this mutation on the protein function.
PCNA (Proliferating Cell nuclear antigen) is a key protein in the regulation of many cellular processes, mainly linked to nucleic acid metabolism; it is considered a processivity factor for the DNA polymerases delta and epsilon, playing an important role in DNA replication; it is also involved in many other pathways, such as Okazaki fragment processing, DNA repair and translesion DNA synthesis, chromatin remodelling, DNA methylation and cell cycle regulation. Since PCNA does not possess any enzymatic activity, its functions are carried out through the interaction with many other proteins working in the different pathways mentioned before. Because of its presence in several intracellular mechanisms and because of its numerous protein interactions, PCNA is also involved in several serious human pathologies such as Systemic Lupus Erythematosus (SLE) and Cancer.The involvement of PCNA in such pathologies underlie the importance of performing further studies on this protein. Particularly important are the studies of PCNA gene mutations.The mouse transgenic technology, that allows the study of the phenotypic effect of gene modification in vivo, represents a great tool for the functional study of gene mutations. This technology can be used for the creation of knock-out mutants (with the aim to either delete part of the DNA sequence or insert irrelevant DNA sequence information to disrupt the expression of a specific genetic locus), or knock-in mutants (aimed to alter the genetic locus of interest via a substitution of DNA sequences or by the addition of a sequence that is not normally found on that genetic locus). One crucial step of this procedure is represented by the screening and the identification of recombinant ES cells. This screening must be not only precise and specific, but also simple. The method of choice for the generation of the knock-in ES cells was the Cre-LoxP system and vector that was composed of a positive selection marker cassette (Neor) and a construct containing a point mutation in the Pcna coding sequence, all flanked by loxP sites. My thesis is about genomic analysis of mouse embryonic stem (ES) cell lines bearing Pcna mutations and my work wants to provide a valid and precise screening strategy that can be applied for the selection of the recombinant ES cells following the knock-in procedure. The strategy developed is based on two main methods: screening by PCR analysis and screening by Southern blot analysis. The transgenic mouse created using this modified ES cells will allow the analysis of the mutated Pcna gene, providing a new model for study the impact of this mutation on the protein function.
Genomic analysis of mouse embryonic stem (ES) cell lines bearing Pcna mutations
MINATO, ADELAIDE
2015/2016
Abstract
PCNA (Proliferating Cell nuclear antigen) is a key protein in the regulation of many cellular processes, mainly linked to nucleic acid metabolism; it is considered a processivity factor for the DNA polymerases delta and epsilon, playing an important role in DNA replication; it is also involved in many other pathways, such as Okazaki fragment processing, DNA repair and translesion DNA synthesis, chromatin remodelling, DNA methylation and cell cycle regulation. Since PCNA does not possess any enzymatic activity, its functions are carried out through the interaction with many other proteins working in the different pathways mentioned before. Because of its presence in several intracellular mechanisms and because of its numerous protein interactions, PCNA is also involved in several serious human pathologies such as Systemic Lupus Erythematosus (SLE) and Cancer.The involvement of PCNA in such pathologies underlie the importance of performing further studies on this protein. Particularly important are the studies of PCNA gene mutations.The mouse transgenic technology, that allows the study of the phenotypic effect of gene modification in vivo, represents a great tool for the functional study of gene mutations. This technology can be used for the creation of knock-out mutants (with the aim to either delete part of the DNA sequence or insert irrelevant DNA sequence information to disrupt the expression of a specific genetic locus), or knock-in mutants (aimed to alter the genetic locus of interest via a substitution of DNA sequences or by the addition of a sequence that is not normally found on that genetic locus). One crucial step of this procedure is represented by the screening and the identification of recombinant ES cells. This screening must not only precise and specific, but also simple. The method of choice for the generation of the knock-in ES cells was the Cre-LoxP system and vector that was composed of a positive selection marker cassette (Neor) and a construct containing a point mutation in the Pcna coding sequence, all flanked by loxP sites. My thesis is about genomic analysis of mouse embryonic stem (ES) cell lines bearing Pcna mutations and my work wants to provide a valid and precise screening strategy that can be applied for the selection of the recombinant ES cells following the knock-in procedure. The strategy developed is based on two main methods: screening by PCR analysis and screening by Southern blot analysis. The transgenic mouse created using this modified ES cells will allow the analysis of the mutated Pcna gene, providing a new model for study the impact of this mutation on the protein function.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/22273