The generation of a murine mesangial cell line deficient in AE2 expression: Introduction of human AE1 into a murine cell line

Persistent Link:
http://hdl.handle.net/10150/290615
Title:
The generation of a murine mesangial cell line deficient in AE2 expression: Introduction of human AE1 into a murine cell line
Author:
Wyant, Timothy Lewis, 1965-
Issue Date:
1996
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:
Senescent cell antigen (SCA) is an antigen that was first identified on human erythrocytes. The formation of SCA is a normal cellular process which marks cells as old and/or damaged. This has been clearly demonstrated in the erythrocyte systems and is believed to occur in other cell types. SCA is formed from the breakdown of the major anion transport protein (Anion Exchanger, AE, or band 3). The mechanism for the formation of SCA is currently not understood. Research into the formation of SCA has been hampered by lack of in vitro models that allow the manipulation of the AE protein. Here, we describe the formation of two cell lines and a cell clone which may assist in the elucidation of the mechanisms of SCA formation by allowing AE manipulation. The murine mesangial Mes-13 cell line was chosen as the parental cell line. Initially, it was determined that Mes-13 cells express the Anion Exchanger 2 (AE2) form of AE. A cell line deficient in the expression of the AE2 protein was generated using an AE2 RNA antisense construct transfected into the Mes-13 cells. The expression of the AE2 protein was reduced by greater than 60 percent in one of the cell clones (A-8). A second cell expressing human AE1 was generated. Human AE1 was cloned using polymerase chain reaction from reticulocyte cDNA and placed into an expression vector. This vector, called Repbd3, was transfected into the Mes-13 cells and the A-8 clone. A 90 kD protein and a 45 kD protein reactive with anti-AE1 antibodies were shown to be expressed in the Mes-13 cells transfected with the Repbd3 construct. Only the 45 kD version was observed in a pooled A-8 cell line. In individually cloned A-8 cells transfected with the Repbd3 vector, expression of the 45 kD and 90 kD proteins were observed at low levels. The cells lines and vectors generated in this work will greatly assist in the understanding of the AE proteins, the elucidation of the mechanisms involved in the formation of SCA, and set up a system to determine residues critical for SCA formation by site-directed mutagenesis of human AE1.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Biology, Cell.; Health Sciences, Immunology.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Microbiology and Immunology
Degree Grantor:
University of Arizona
Advisor:
Kay, Marguerite M. B.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titleThe generation of a murine mesangial cell line deficient in AE2 expression: Introduction of human AE1 into a murine cell lineen_US
dc.creatorWyant, Timothy Lewis, 1965-en_US
dc.contributor.authorWyant, Timothy Lewis, 1965-en_US
dc.date.issued1996en_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.abstractSenescent cell antigen (SCA) is an antigen that was first identified on human erythrocytes. The formation of SCA is a normal cellular process which marks cells as old and/or damaged. This has been clearly demonstrated in the erythrocyte systems and is believed to occur in other cell types. SCA is formed from the breakdown of the major anion transport protein (Anion Exchanger, AE, or band 3). The mechanism for the formation of SCA is currently not understood. Research into the formation of SCA has been hampered by lack of in vitro models that allow the manipulation of the AE protein. Here, we describe the formation of two cell lines and a cell clone which may assist in the elucidation of the mechanisms of SCA formation by allowing AE manipulation. The murine mesangial Mes-13 cell line was chosen as the parental cell line. Initially, it was determined that Mes-13 cells express the Anion Exchanger 2 (AE2) form of AE. A cell line deficient in the expression of the AE2 protein was generated using an AE2 RNA antisense construct transfected into the Mes-13 cells. The expression of the AE2 protein was reduced by greater than 60 percent in one of the cell clones (A-8). A second cell expressing human AE1 was generated. Human AE1 was cloned using polymerase chain reaction from reticulocyte cDNA and placed into an expression vector. This vector, called Repbd3, was transfected into the Mes-13 cells and the A-8 clone. A 90 kD protein and a 45 kD protein reactive with anti-AE1 antibodies were shown to be expressed in the Mes-13 cells transfected with the Repbd3 construct. Only the 45 kD version was observed in a pooled A-8 cell line. In individually cloned A-8 cells transfected with the Repbd3 vector, expression of the 45 kD and 90 kD proteins were observed at low levels. The cells lines and vectors generated in this work will greatly assist in the understanding of the AE proteins, the elucidation of the mechanisms involved in the formation of SCA, and set up a system to determine residues critical for SCA formation by site-directed mutagenesis of human AE1.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectBiology, Cell.en_US
dc.subjectHealth Sciences, Immunology.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineMicrobiology and Immunologyen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorKay, Marguerite M. B.en_US
dc.identifier.proquest9713375en_US
dc.identifier.bibrecord.b34372064en_US
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