Author/Editor		Mogilnicki, Lea
Title		Zasledovanje sinteze in gibanja beljakovin v sesalskih celicah s fluorescenčno beljakovino EGFP
Type		monografija
Place		Ljubljana
Publisher		Medicinska fakulteta
Publication year		2000
Volume		str. 42
Language		slo
Abstract		INTRODUCTION The process of exocytosis can be studied by following the movement vesicles under confocal microscope. One way of fluorescently labelling the vesicles is by attaching EGFP (green fluorescent protein) to the membrane protein native to vesicles, e.g. to synaptotagmin. Previous experiments have shown that when the fusion proteins synaptotagmin-EGFP are expressed from the constitutive promoter, the fluorescent signal is spread over the whole cell. In order to be able to follow the vesicle movement, one has to control the synthesis of the fluorescent protein. The possibility to regulate the protein synthesis is based on new technologies based on cell culturing and genetics. It is hoped this techniques will revolutionise the medical research as well as will provide transplants, recombinant drugs and advance techniques ofi gene therapy. In basic research, the regulation of protein synthesis is used when the protein expressed is toxic for cells, has to be expressed in a narrow time window or when producing transgenic animals. The use of systems for regulated expression to produce fluorescent proteins have not been reported so far. AIMS As no data were available on the regulated expression of fluorescent proteins, we decided to investigate whether tetracycline-based Tet-off system could be suitable for expresing EGFP. As the fluorescence of EGFP is the sum of fluorescences of individual molecules (when there are no energy losses), our first task was to find out whether one can induce EGFP enough to observe the cell fluorescence. HYPOTHESES Our hypotheses were: 1. Tet-off system can be used for the expression ofi fluorescent proteins 2. After triggering the synthesis, enough EGFP will be synthesised to detect tluorescent cells underthe microscope 3. Synthesis of EGFP can be detected by following the tluorescence in an individual cell under confocal microscope. (Abstract truncated at 2000 characters).
Descriptors		PROTEINS FLUORESCENT ANTIBODY TECHNIQUE EXOCYTOSIS GENE EXPRESSION REGULATION MAMMALS CELLS, CULTURED MICROSCOPY, CONFOCAL GENETIC VECTORS POLYMERASE CHAIN REACTION