Characterisation of VMO1 in Human Tissues
Crossan, H. C. (2014). Characterisation of VMO1 in Human Tissues (Thesis, Master of Science (MSc)). University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/8792
Permanent Research Commons link: https://hdl.handle.net/10289/8792
The Vitelline membrane outer layer protein 1 (VMO1) was first identified inthe Vitelline membrane of the chicken egg. This membrane almost entirelyprotein that separates the egg white from the yolk in amniotic eggs andacts as the last defensive barrier between pathogens and the egg yolkwith the developing embryo. Since its discovery in the vitelline membrane,VMO1 has been isolated from a number of different tissues and animals.The one that would appear to have the largest impact on human healthwould be its discovery in the Reissner’s membrane of mice, whoseauditory system has high structural homology to humans. The Reissner’smembrane maintains the electrolyte level in the compartments of the innerear as well as the resting electrochemical potential which are both criticalfor correct hearing function.The main aims of the research undertaken in this thesis was to 1)determine the tissues in which VMO1 is or is not expressed in humansand 2) create a genetically modified expression clone of human VMO1 touse in further downstream protein-protein interaction studies. Ourhypothesis is that we can amplify VMO1 from human cell lines based onavailable bioinformatics data.The first objective was to determine the gene expression of VMO1 inmultiple human cell lines. RNA was extracted from three commercial celllines (list) and the integrity of RNA was confirmed by agarose gelelectrophoresis by the observation of two rRNA bands. From this cDNAAbstractiiiwas made, and PCR analysis performed to identify the expression, or not, of VMO1 in these tissues. Successful amplification of VMO1 from lung cells (A549) lead to the creation of a human VMO1 expression clone, by transforming competent Escherichia coli cells with ligated VMO-1 insert/pPLUG vector. White colonies were selected for DNA extraction and confirmed as positive using colony PCR and agarose gel electrophoresis. DNA seuqencing of the positive clone confirmed the nucleotide sequence as VMO1.The second objective was to validate the commercial antibody for human VMO1 using physiological and immunohistochemical methodology. The immunohistochemistry data suggests that the VMO1 protein is a secreted protein since signal was detected in the P5 mouse inner ear and mouse adult lung. However, multiple bands were observed in the western blot. Futher investigation is required to validate the VMO1 antibody to demonstrate that it is indeed specific to recognising its target epitope. The production of recombinant VMO1 protein would be benefical to address this question.
University of Waikato
All items in Research Commons are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
- Masters Degree Theses