Preparation of Fluorescent Tissue Factor Fusion Protein Constructs for Stable Transfection of Cell Lines
Mentor 1
Julie Oliver
Location
Union Wisconsin Room
Start Date
24-4-2015 10:30 AM
End Date
24-4-2015 11:45 AM
Description
Tissue factor is the biologically relevant initiator of blood coagulation. Coagulation begins when blood comes into contact with tissue factor-bearing cells when the vasculature is damaged. However, full exposure of tissue factor function, or decryption, requires a secondary cell activation event. The nature of this cell activation event is a matter of debate. One hypothesis suggests that tissue factor molecules in resting cells are dimerized, leading to encryption of activity by physical blockade of substrate binding sites. Cell activation would be expected to separate the dimers into monomers, exposing maximal substrate binding and full function. We will test the hypothesis by generating expression vectors containing full length human tissue factor cDNA fused with the sequence for either yellow fluorescent protein (YFP) or green fluorescent protein (GFP).When co-expressed in mammalian cells, the fluorescent proteins will act as a matched pair for Förster resonance energy transfer (FRET) analysis. FRET signaling can be used to analyze oligomerization of molecules. Two experimental approaches will be undertaken: (i.) design and use of polymerase chain reaction (PCR) primer pairs to amplify the tissue factor sequence from an already existing plasmid, and KpnI or XbaI (or alternatively SacI and XbaI) restriction sites to be used in the cloning of tissue factor sequence into existing expression vectors containing GFP or YFP that will be in-frame with the PCR product insert, and (ii.) cut the YFP and GFP sequences out of their respective plasmids at HindIII and SacI restriction sites, and ligate at those restriction sites upstream and in-frame with the tissue factor sequence in its existing expression vector. If the tissue factor dimerization hypothesis is shown to be correct and to play a significant role the initiation of coagulation, site-directed mutagenesis will be undertaken. Specific amino acid substitutions could then be used to examine the effect of modification on tissue factor function. These experiments will help more fully understand the role of tissue factor in health and disease.
Preparation of Fluorescent Tissue Factor Fusion Protein Constructs for Stable Transfection of Cell Lines
Union Wisconsin Room
Tissue factor is the biologically relevant initiator of blood coagulation. Coagulation begins when blood comes into contact with tissue factor-bearing cells when the vasculature is damaged. However, full exposure of tissue factor function, or decryption, requires a secondary cell activation event. The nature of this cell activation event is a matter of debate. One hypothesis suggests that tissue factor molecules in resting cells are dimerized, leading to encryption of activity by physical blockade of substrate binding sites. Cell activation would be expected to separate the dimers into monomers, exposing maximal substrate binding and full function. We will test the hypothesis by generating expression vectors containing full length human tissue factor cDNA fused with the sequence for either yellow fluorescent protein (YFP) or green fluorescent protein (GFP).When co-expressed in mammalian cells, the fluorescent proteins will act as a matched pair for Förster resonance energy transfer (FRET) analysis. FRET signaling can be used to analyze oligomerization of molecules. Two experimental approaches will be undertaken: (i.) design and use of polymerase chain reaction (PCR) primer pairs to amplify the tissue factor sequence from an already existing plasmid, and KpnI or XbaI (or alternatively SacI and XbaI) restriction sites to be used in the cloning of tissue factor sequence into existing expression vectors containing GFP or YFP that will be in-frame with the PCR product insert, and (ii.) cut the YFP and GFP sequences out of their respective plasmids at HindIII and SacI restriction sites, and ligate at those restriction sites upstream and in-frame with the tissue factor sequence in its existing expression vector. If the tissue factor dimerization hypothesis is shown to be correct and to play a significant role the initiation of coagulation, site-directed mutagenesis will be undertaken. Specific amino acid substitutions could then be used to examine the effect of modification on tissue factor function. These experiments will help more fully understand the role of tissue factor in health and disease.
