Persistent Link:
http://hdl.handle.net/10150/185626
Title:
A NOVEL GENE TRANSFER SYSTEM FOR MAMMALIAN CELLS.
Author:
SLILATY, STEVE N.
Issue Date:
1983
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Productive infection of mouse cells with polyoma virus yields mainly two types of particles: Complete virions and empty capsids. Empty polyoma capsids have been shown to be capable of interacting with DNA, in vitro, to form what has been referred to as polyoma-like particles (PLP). The particles are stable in high concentrations of salt and contain DNA protected by the capsid against the action of pancreatic DNase. The development of PLP into a gene transfer vehicle is the subject of the investigations described in the present dissertation. The approach has been to first, characterize the process of PLP formation and second, determine whether the genetic information contained in a specific DNA fragment and assembled into PLP in vitro can be transferred to cells and subsequently be expressed. In terms of PLP characteristics, the experimental results described in this dissertation show that the DNA extracted from PLP is heterogeneous in size. It has a mean molecular weight of 1.2 x 10⁶ with a standard deviation of ±0.5 x 10⁶. In addition, analysis of PLP DNA with restriction endonucleases revealed that a specific primary sequence or higher order structure is not required for PLP formation. Either linear, circular or supercoiled polyoma DNA, as well as, single-stranded DNA, rRNA and the synthetic homopolymers poly(dA).poly(dT) and poly(dG).poly(dC) can be used for PLP formation. Transfer of genetic information by PLP has been accomplished by using a restriction fragment containing the transforming sequences of polyoma DNA as a model gene. This fragment of polyoma DNA, which consists of 1,831 base pairs (approximately 1.2 x 10⁶ daltons) and extends clockwise from the BclI site to the EcoRI site on the conventional polyoma map, causes the induction of the transformed phenotype in rat cells grown in culture. Infection of rat F111 cells by PLP, containing this DNA fragment, results in DNA-mediated oncogenic transformation of the cells as indicated by the formation of dense foci. This gene transfer activity of PLP is shown to be 50 to 150 times more efficient than the widely used calcium phosphate coprecipitation method of introducing DNA into mammalian cells.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Polyomaviruses -- Genetics.; Genetic engineering.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Cellular and Developmental Biology; Graduate College
Degree Grantor:
University of Arizona

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleA NOVEL GENE TRANSFER SYSTEM FOR MAMMALIAN CELLS.en_US
dc.creatorSLILATY, STEVE N.en_US
dc.contributor.authorSLILATY, STEVE N.en_US
dc.date.issued1983en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractProductive infection of mouse cells with polyoma virus yields mainly two types of particles: Complete virions and empty capsids. Empty polyoma capsids have been shown to be capable of interacting with DNA, in vitro, to form what has been referred to as polyoma-like particles (PLP). The particles are stable in high concentrations of salt and contain DNA protected by the capsid against the action of pancreatic DNase. The development of PLP into a gene transfer vehicle is the subject of the investigations described in the present dissertation. The approach has been to first, characterize the process of PLP formation and second, determine whether the genetic information contained in a specific DNA fragment and assembled into PLP in vitro can be transferred to cells and subsequently be expressed. In terms of PLP characteristics, the experimental results described in this dissertation show that the DNA extracted from PLP is heterogeneous in size. It has a mean molecular weight of 1.2 x 10⁶ with a standard deviation of ±0.5 x 10⁶. In addition, analysis of PLP DNA with restriction endonucleases revealed that a specific primary sequence or higher order structure is not required for PLP formation. Either linear, circular or supercoiled polyoma DNA, as well as, single-stranded DNA, rRNA and the synthetic homopolymers poly(dA).poly(dT) and poly(dG).poly(dC) can be used for PLP formation. Transfer of genetic information by PLP has been accomplished by using a restriction fragment containing the transforming sequences of polyoma DNA as a model gene. This fragment of polyoma DNA, which consists of 1,831 base pairs (approximately 1.2 x 10⁶ daltons) and extends clockwise from the BclI site to the EcoRI site on the conventional polyoma map, causes the induction of the transformed phenotype in rat cells grown in culture. Infection of rat F111 cells by PLP, containing this DNA fragment, results in DNA-mediated oncogenic transformation of the cells as indicated by the formation of dense foci. This gene transfer activity of PLP is shown to be 50 to 150 times more efficient than the widely used calcium phosphate coprecipitation method of introducing DNA into mammalian cells.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectPolyomaviruses -- Genetics.en_US
dc.subjectGenetic engineering.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineCellular and Developmental Biologyen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.committeememberHewlett, Martyen_US
dc.contributor.committeememberKeck, Konraden_US
dc.contributor.committeememberLaw, Johnen_US
dc.contributor.committeememberSalemme, Rayen_US
dc.identifier.proquest8311418en_US
dc.identifier.oclc688339895en_US
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