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ID naloge: 84 Letnik: 2000 Predmet: biologija celice
Zasledovanje sinteze in gibanja beljakovin v sesalskih celicah s fluorescencno beljakovino EGFP
Avtor: Lea Mogilnicki
Mentor: znanst.sod.dr. Irina Milisav
Eden izmed nacinov študija procesa eksocitoze v živi celici je z zasledovanjem premikanja membranskih mešickov pod konfolkalnim mikroskopom. Mešicke lahko fluorescencno oznacimo' z izdelavo fuzijske beljakovine sy-EGFP (med membransko beljakovino v mešicku sinaptotagmin in fluorescencno beljakovino EGFP). Predhodni poskusi so pokazali, da po vnosu take beljakovine fluorescencni signal preplavi celotno celico; za zasledovanje potovanja mešickov, bi morali nadzorovati sintezo tluorescencne beljakovine.
Možnost regulacije sinteze beljakovin v sesalskih celicah je novo odkritje, ki obeta veliko možnih nacinov uporabe v medicini (na primer: za izdelavo nekaterih rekombinantnih zdravil, pridobivanje tkiv in organov za presadke, pri genski terapiji), pri raziskovalnem delu pa pri izdelavi transgenih živali, študiju beljakovin, ki se morajo pojaviti v tocno dolocenem casu ali so toksicne za celico. V literaturi nismo zasledili, da bi bili sistemi za regulacijo ekspresije beljakovin v sesalskih celicah do zdaj uporabljeni za nadzorovanje sinteze fluorescencnih beljakovin.
NAMEN
Zaradi potrebe po regulaciji sinteze fluorescencne beljakovine EGFP in pomanjkanja podatkov o uporabnosti sistemov za regulirano ekspresijo fluorescencnih beljakovin, smo sklenili preveriti ali je sistem regulacije s tetraciklinom, Tet-off, primeren za ekspresijo zelene fluorescencne beljakovine, EGFP. Ker je oddana svetloba vsota fluorescence posameznih molekul EGFP (z upoštevanjem izgub), se mora v celici v primernem casu sintetizirati dovolj molekul beljakovine, da bi lahko zaznali fluorescenco.
HIPOTEZE
Postavili smo naslednje hipoteze:
1 . sistem za regulirano ekspresijo beljakovin je primeren za ekspresijo fluorescencne beljakovine EGFP.
2. s sistemom Tet-off se izrazi dovolj fluorescencne beljakovine, da bomo lahko zaznali povecanje števila fluorescencnih celic pod mikroskopom
3. sintezo EGFP v posameznih celicah se lahko v realnem casu spremlja z zasledovanjem fluorescence s konfokalnim mikroskopom
4. regulirana ekspresija EGFP lahko omogoci zasledovanje potovanja beljakovin z mesta sinteze po celici
METODE
Da bi lahko spremljali izražanje fluorescencne beljakovine EGFP, smo morali izvesti tri sklope poskusov: (1) izdelavo konstruktov za regulirano ekspresijo beljakovin (s tehnikami genske tehnologije), (2) pridobivanje celicne linije PC12, ki omogoca regulirano sintezo EGFP (gojenje celicnih linij, vnos DNA v sesalske celice) in (3) optimizacija pogojev ter zasledovanje EGFP v celicah (merjenje aktivnosti encima luciferaze, spremljanje tluorescence s fluorescencnim in konfokalnim mikroskopom).
REZULTATI
Pripravili smo konslrukt za regulirano ekspresijo EGFP, pTRE2-EGFP in ga vnašali v celice podganje celicne linije PC12 Tet-off. Tako smo pridobili sistem za izražanje EGFP, reguliran s koncentracijo tetraciklina. Dodajanje 2 µg/ml tetraciklina v celicni medij, ni vplivalo na hitrost sinteze beljakovin, ki niso bile regulirane s tetraciklinom. Celice PC12 so rasle v skupkih, vendar število celic v skupku ni vplivalo na delež fluorescencnih celic. Po sproženju sinteze EGFP z odvzemom tetraciklina, se je v 30. h delež fluorescencnih celic izenacil z deležem v kontrolnih vzorcih, kjer je bila ves cas omogocena sinteza EGFP. Pri tem se je delež fluorescencnih celic po sproženju sinteze poprecno povecal za 5-krat, medtem ko se je delež fluorescencnih celic v kontroli v istem casu le podvojil. Fluorescenco EGFP v posamezni celici smo lahko zasledovali tudi s konfokalnim mikroskopom, vendar iz podatkov ni bilo mogoce analizirati potovanja EGFP po celici.
ZAKLJUCKI
Sistem za regulirano ekspresijo beljakovin je primeren za ekspresijo fluorescencne beljakovine EGFP. Po sproženju sinteze smo izrazili dovolj beljakovine, da smo zaznali povecanje fluorescence skupine celic, kakor tudi povecanje fluorescence v posamezni celici. Analiza fluorescencnih signalov posameznih celic ni omogocila spremljanje beljakovine po celici, zaradi nihanja celic med opazovanjem iz konfokalne ravnine. Nadaljnji poskusi bodo usmerjeni v reševanje teh težav.
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[Abstract / English version]
Folowing protein synthesis and the vesicle movement in mamallian cells with green fluorescent protein EGFP
Author: Lea Mogilnicki
Mentor: znanst.sod.dr. Irina Milisav
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 synaptot agmin. Previous experiments have shown that when the fusion proteins synaptotagmin-EGFP are expressed from the constitutive promoter, the t7uorescent 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 fi7uorescent 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 of 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 t7uorescent proteins, we decided to investigate whether tetracycline-based Tet-off system could be suitable for expressing 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 of fluorescent proteins
2. After triggering the synthesis, enough EGFP will be synthesised to detect fluorescent cells under the microscope
3. Synthesis of EGFP can be detected by following the fluorescence in an individual cell under confocal microscope
4. Regulated expression of EGFP enables tracing of the protein within the cells. METHODS
To follow the filuorescence of EGFP, we had to ( 1 ) construct the DNA, which enables regulated expression of EGFP (with techniques of gene technology), (2) prepare the cell culture and transfect the cells with the EGFP construct, (3) optimise the expression conditions and follow up the fluorescence by fluorescent and confocal microscopes.
RESULTS
Firstly, we cloned the construct for regulated expression of EGFP, pTRE2-EGFP. Introducing the plasmid pTRE2-EGFP into PCl2 Tet-off cells enabled us to regulate the expression of EGFP by varying the concentration of tetracycline. Preliminary results showed that the protein synthesis was inhibited by adding 2 µg/ml tetracycline into the rnedium. PC 12 cells tend to arrow in bundles; the number of cells in a bundle had no effect on percentage of fluorescent cells. Thirty hours after induction of protein synthesis, the percentage of fluorescent cells reached the level equal to that in the control sample (grown through without tetracycline). The percentage of fluorescent cells after induction of EGFP synthesis increased 5-fold, while the percentage of fluorescent cells in a control experiment increased only by 2-fold during the satne period. After induction of EGFP synthesis, enough protein was made to detect the fluorescence within the individual cells, however, we were not able to trace the movement of EGFP molecules within the individual cells.
CONCLUSIONS
The system fcn- regulated expression of proteins is suitable for the regulated expression of EGFP. It is possible to produce enough fluorescent protein to detect the filuorescence of a group of cells and within individual cells. Despite that we were not able to follow the migration of individual molecules within the cells due to frequent movements (rotations) of the cells. That will be addressed in future studies.
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