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Title: The use of gene disruption to study the cytoskeletal proteins talin and vinculin
Author: Priddle, Helen
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 1996
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Talin and vinculin are cytoskeletal proteins co-localising with the integrin family of adhesion receptors at sites of adhesion to the extracellular matrix. To investigate the role of these proteins in cell adhesion, gene disruption by homologous recombination has been used in an attempt to produce cells deficient in talin or vinculin. A C129 mouse embryonic stem (ES) cell ? library was screened with a 5' mouse talin cDNA and talin genomic clones isolated. A promoterless neo gene was inserted into the genomic DNA within the first two coding exons, forming a talin gene disruption vector. This vector failed to confer G418 resistance to ES cells, possibly due to the use of a neo gene containing a mutation which reduces the phosphotransferase activity of the encoded protein. A further vector was constructed using the same section of talin genomic DNA with a wild type neo gene driven by a PGK promoter. A tk cassette was included for selection against random integration. This vector was used successfully to isolate ES cells with one allele of the talin gene disrupted. However, talin levels were not reduced in these cells. This cell line will be useful to produce talin deficient cells or mice for the study of talin function. A vinculin gene targeting vector containing a tk negative selection marker and a neo gene driven by a PGK promoter was used unsuccessfully to disrupt the vinculin gene in ES cells. An alternative vector was constructed with a promoterless neo to reduce random integration, but this approach was also unsuccessful. The original vector used the mutant version of neo. The use of a disruption vector employing a wild type neo cassette has since led to the disruption of the vinculin gene. Wistar Furth rats have a point mutation (pro 1176 thr) in the talin gene. The mutation was introduced into a talin cDNA encoding residues 565-1328. The mutant and wild type cDNAs were expressed as GST-fusion proteins. The vinculin- and actin- binding activities of the expressed fusion proteins were assayed in vitro. The mutation did not prevent the binding of the talin polypeptide to vinculin or actin and the mutant polypeptide was still able to localise to focal adhesions when microinjected into fibroblasts.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available