Functional Characterisation of Syndecan, a heparan sulpahte proteoglycan, in Slit/Robo signalling

Abstract

Introduction__________________________________________________________ 1.1 Heparan Sulphate Proteoglycans (HSPGs) ___________________________________ 1.2 Roles of HSPGs in Signalling ______________________________________________ 1.3 The Drosophila ventral midline____________________________________________ 1.4 Slit/Robo signalling mediates axon guidance_________________________________ 1.5 Slit/Robo signalling mediates muscle patterning______________________________ 1.6 Syndecan regulates Slit/Robo signalling ____________________________________ 1.7 Domain structure of Syndecan ____________________________________________1.7.1 Cytoplasmic domain of Syndecan_______________________________________________ 1.7.2 Transmembrane domain of Syndecan ____________________________________________ 1.7.3 Extracellular domain of Syndecan ______________________________________________ 1.8 Mechanistic analysis of Syndecan function in Slit/Robo signalling _______________ 2 Materials and Methods ________________________________________________ 2.1 Molecular Biology ______________________________________________________ 2.1.1 Polymerase Chain Reaction ___________________________________________________ 2.1.2 DNA Restriction ____________________________________________________________ 2.1.3 DNA extraction from agarose gel _______________________________________________ 2.1.4 Dephosphorylation of vector DNA ______________________________________________ 2.1.5 DNA Ligation ______________________________________________________________ 2.1.6 Preparation of electro-competent Escherichia coli (E.coli) cells _______________________ 2.1.7 Preparation of chemical-competent E.coli cells ____________________________________ 2.1.8 Transformation of electro-competent E.coli cells ___________________________________ 2.1.9 Transformation of chemical-competent E.coli cells _________________________________ 2.1.10 Plasmid DNA purification____________________________________________________ 2.1.11 DNA Sequencing __________________________________________________________ 2.1.12 Site-directed mutagenesis ____________________________________________________ 2.1.13 DNA preparation for Embryo Injections_________________________________________ 2.2 Fly techniques __________________________________________________________ 2.2.1 Maintenance of flies _________________________________________________________ 2.2.2 Generation of stable transgenic fly lines __________________________________________ 2.2.3 Ectopic gene expression via the UAS/GAL4 system ________________________________ 2.2.4 Fixation of embryos _________________________________________________________ 2.2.5 Antibody (Ab) staining of embryos _____________________________________________ 2.2.6 Fluorescence microscopy _____________________________________________________ 2.3 Cell culture based techniques _____________________________________________ 2.3.1 Maintenance of cells _________________________________________________________ 2.3.2 Transfection of cells _____________________________________...