Engineering of a Collagen IV::Gfp Fusion Protein Using Polymerase Chain Reaction
Date of Graduation
Master of Science in Cell and Molecular Biology
Collagen IV, Caenorhabditis elegans, polymerase chain reaction, gene fusion, GFP
Medical Molecular Biology
Collagen type IV, a component of the basement membrane, has been linked to several human diseases. These diseases include the hereditary disease Alport's syndrome and the autoimmune disease Goodpasture syndrome. The nematode Caenorhabditis elegans has been shown to be a good model system in which to study the function and location of collagen type IV in vivo. Previously, these studies have been accomplished by immunofluorescents staining requiring fixation and sacrificing of the worms which can introduce artifacts. In order to fully study the true localization and dynamics of collagen type IV the worms should be living. This can be accomplished by the production of a collagen type IV protein tagged with a reporter gene such as green fluorescent protein (GFP). These experiments focused on the production of a C. elegans collagen type IV::GFP fusion protein that would enable collagen type IV to be visualized in vivo. Attempts at production of the fusion gene used a specialized PCR technique, overlap extension PCR, to enable the production of long PCR products. The technique used heterologous primers to produce overhangs on the emb-9 gene that would have been used to insert the gfp gene in the desired location. The location within the emb-9 gene that was focused on was near the middle of a 13 amino acid interruption in the Gly-X-Y region near the 7S domain. the resulting fusion gene would have had the complete gfp gene inserted between nucleotides 1842 and 1843. Successful PCR attempts resulted in the amplification of the unspliced gfo gene and 3' emb-9 fragment. Unsuccessful attempts to amplify the 5' emb-9 fragment resulted in the discovery of the possible formation of primer dimers inhibiting the production of the collagen type IV::GFP fusion protein.
© Courtney Elizabeth Eaves
Eaves, Courtney Elizabeth, "Engineering of a Collagen IV::Gfp Fusion Protein Using Polymerase Chain Reaction" (2007). MSU Graduate Theses. 2836.