Thesis Title

Production Of A Functional Collagen Type IV:: GFP Fusion Protein Reporter Construct In Caenorhabditis Elegans

Date of Graduation

Fall 2004

Degree

Master of Science in Cell and Molecular Biology

Department

Biomedical Sciences

Committee Chair

Colette Witkowski

Keywords

Collagen IV, C. elegans, PCR, gene fusion, GFP

Subject Categories

Medical Molecular Biology

Abstract

Collagen type IV is one of the most abundant structural molecules in the basement membrane and mutations have been indicated in several human diseases. Collagen type IV has previously been studied in C. elegans using immunofluorescent staining techniques that involve fixation, which kills the animals, or indirectly in live animals using reporter constructs of proteins that are in close proximity to the basement membrane. It is of interest to study the localization, structure, and dynamics of this macromolecule in living worms to understand the function by directly tagging the collagen type IV molecule with a green fluorescent protein (GFP) reporter that allows the protein to be visualized in vivo. Production of the reporter gene was undertaken by using a specialized polymerase chain reaction (PCR) technique to fuse GFP to collagen type IV. The technique involves sequential PCR reactions, some of which use heterologous primers to create an overhang on the amplified portions of the emb-9 gene, homologous to the gfp gene. During subsequent PCR amplifications the overlapping sites produced in the previous reactions were fused producing a product containing the necessary sequence information for translation of a protein containing the functional properties of both collagen type IV and GFP. Several sites near the N-terminus of the collagen type IV molecule were selected as possible fusion sites. An asparagine-linked glycosylation site identified as a location for possible fusion was examined in this study. After optimization, the final PCR product was microinjected along with rol-6, a dominant selectable marker, into the synctial gonad of C. elegans heterozygous for a collagen type IV null mutation which causes embryonic lethality. Production of a functional fusion protein was not yet indicated by rescue of the homozygous recessive class with roller and GFP phenotypes. F1 transgenic worms were obtained and one F2 transgenic worm has been isolated so far, but was sterile and could not be used to establish a strain.

Copyright

© David K. Stone

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