Promoter. Transcription factor Green Fluorescent Protein (GFP) The green fluorescent protein is found in a jellyfish that lives in the.

Presentación del tema: "Promoter. Transcription factor Green Fluorescent Protein (GFP) The green fluorescent protein is found in a jellyfish that lives in the."— Transcripción de la presentación:

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11 Green Fluorescent Protein (GFP) The green fluorescent protein is found in a jellyfish that lives in the cold waters of the north Pacific. The jellyfish contains a bioluminescent protein-- aequorin--that emits blue light, and a green fluorescent protein that converts this light to green light, which is what we actually see when the jellyfish lights up. The protein absorbs either blue or ultraviolet light, and emits it as lower-energy green light. So What? You might be saying: who cares about this obscure little green protein from a jellyfish? It turns out that GFP is amazingly useful in scientific research, because it allows us to look directly into the inner workings of cells. It is easy to find out where GFP is at any given time: you just have to shine ultraviolet light, and any GFP will glow bright green. So here is the trick: you attach the GFP to any object that you are interested in watching. For instance, you can attach it to a virus. Then, as the virus spreads through the host, you can watch the spread by following the green glow. Or, you can attach it to a protein, and watch through the microscope as it moves around inside cells.

12 Ready-Made GFP is a ready-made fluorescent protein, so it is particularly easy to use. Most proteins that deal with light use exotic molecules to capture and release photons. For instance, the opsins in our eyes use retinol to sense light (see the Molecule of the Month on bacteriorhodopsin). These "chromophores" must be built specifically for the task, and carefully incorporated into the proteins. GFP, on the other hand, has all of its own light handling machinery built in, constructed using only amino acids. It has a special sequence of three amino acids: serine-tyrosine-glycine (sometimes, the serine is replaced by the similar threonine). When the protein chain folds, this short segment is buried deep inside the protein. Then, several chemical transformations occur: the glycine forms a chemical bond with the serine, forming a new closed ring, which then spontaneously dehydrates. Finally, over the course of an hour or so, oxygen from the surrounding environment attacks a bond in the tyrosine, forming a new double bond and creating the fluorescent chromophore. Since GFP makes its own chromophore, it is perfect for genetic engineering. You don't have to worry about manipulating any strange chromophores; you simply engineer the cell with the genetic instructions for building the GFP protein, and GFP folds up by itself and starts to glow.

13 DNA mRNA protein transcription translation promoter GFPYour Favorite Gene UV light visible green light GFPYour Favorite Gene

14 nucleus E2F-DP pRB Cyclin D1 accumulation into the nucleus as a key step in cell cycle entry cytoplasm Whi3 CycD1 Cdk4,6 5' CycD1 mRNA cell cycle entry S M G1G1 G2G2 cell cycle exit and differentiation

15 Exogenous CycD1 reproduces similar localization patterns under cycling and G1 arrest conditions in NIH3T3 cells EGFP-CycD1 Nuclei EGFP-CycD1 Nuclei Deprived Cycling Neus Colomina, Maria Ruiz and Martí Aldea (unpublished)

16 Shown here is a time lapse that depicts bi-directional movements of Survival Motor Neuron protein (SMN) fused to GFP. Deletion of exon 7 causes Spinal Muscular Atrophy Zhang et al. J Neurosci 23:6627-6637 (2003) GFP-bound  -actin mRNA was observed in the form of granules (displayed in black) that were distributed throughout the neurites Transport of SMN and actin mRNA granules within neurites

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18 DNA mRNA protein transcription translation peroxisomes 256 lacO sequences LacI-CFP 24 MS2 sequences MS2p-GFP localization CFP-peroxisomal signal (ORF) CFP-peroxisomal signal 24 MS2 sequences

19 Visualizing gene expression in living cells. The movie shows a cell with a stably integrated gene that also contains 256 lac operator repeats. This gene transcribes an RNA that contains both a coding sequence for the cyan fluorescent protein (CFP) protein (with a peroxisome-targeting sequence) and a stretch of MS2 stem-loops. In the beginning of the movie, the gene locus is visible as a result of tagging of the DNA with a CFP–lac-repressor protein. Once transcription is induced from this gene, the locus becomes structurally open and decondenses. The RNAs produced from the gene are tagged with green fluorescent protein (GFP)–MS2 and can be seen accumulating at the transcription site. The RNA is translated in the cytoplasm and at later times post-induction, CFP-labelled peroxisomes are detected. The cell was imaged every 2.5 min for a total of 4 hr and 22.5 min. Janicki et al. Cell 116, 683-698 (2004). LacI-CFP MS2p-GFP CFP-peroxisomal signal

20 La regulació a nivell transcripcional és el mode fonamental de regulació de l’expressió gènica, i ens permet entendre cóm els gens poden donar origen a l’enorme multiplicitat funcional dels organismes pluricel·lulars complexos

21 Durant el desenvolupament es generen diferents combinacions de factors transcripcionals específics que indiquen els camins diversos de diferenciació per a cada cèl·lula Aquest model simple assumeix que la posició en cada divisió activa (o fa aparèixer) uns factors transcripcionals específics diferents Si continuéssim amb l’esquema proposat tindríem més de 10000 tipus cel·lulars diferents amb només 25 factors transcripcionals específics

22 La diferent localització dels mRNAs en una cèl·lula en mitosi pot originar dues cèl·lules filles diferents Mitosi

23 Movie of ASH1 mRNA Particle Movement This four minute, time-lapse movie (clock in seconds) depicts the movement of a particle, detected by a GFP-MS2 chimera bound to ASH1 mRNA, in budding yeast cells (mother on bottom, daughter on top). While the particle in the wildtype yeast strain (left) travels through the bud neck and localizes to the bud tip, the particle in the yeast strain containing a deletion of she1 (right) exhibits minimal displacement and does not leave the mother cell. The particle's peak velocity reaches 440 nm/s. (bar = 2 micrometers) Bertrand et al. Mol Cell 2:437-445 (1998)

24 La diferent localització dels mRNAs en la cèl·lula pot restringir localment l’expressió d’una proteïna a nivell traduccional

25 Accumulation of fluorescent nanos RNA at the oocyte posterior cortex A time-lapse movie of a Drosophila stage-12 egg chamber containing nanos–MS2 RNA that was labelled with MS2–GFP. Fluorescent nanosforms particles as it accumulates at the posterior cortex. Autofluorescent yolk granules are seen passing in the background. Each second represents 23 min of oogenesis. Forrest & Gavis, Curr. Biol. 13, 1159-1168 (2003). nanos mRNA Posterior cortex

26 n Inserció a l’atzar de còpies múltiples d’un gen concret Substitució d’un gen concret per recombinació homòloga 1. INSERCIÓ (transgenic) 3. SUBSTITUCIÓ (knock-in) 2. DELECIÓ (knock-out) Eliminació d’un gen concret per recombinació homòloga 3 tipus de manipulació genètica sobre el DNA

27 Injecció de DNA Manipulació (o teràpia) gènica d’organismes en línia germinal per injecció de DNA normal transgenic mouse for growth hormone gene Injection of DNA into a fertilized egg black skin gene white mouse transgenic (inserció)

28 Manipulació (o teràpia) gènica d’organismes en línia germinal per implantació de cèl·lules precursores embrionàries modificades genèticament Injection of a genetically modified cell into a blastula mosaic gonad mosaic cells X Implantació de cèl·lules knock-out (deleció) knock-in (substitució) white mouse black mouse cell mosaic mouse

29 Com un exemple de teràpia gènica somàtica en fase de proves per al tractament del càncer, s’utilitzen vectors virals per a reintroduir gens supressors de tumors en cèl·lules canceroses, i frenar no solament la seva proliferació sinó també la de cèl·lules veïnes tumor supressor gene transfected cell tumor cell

30 Com un altre exemple de teràpia gènica somàtica, actualment en fase de proves per a tractar les malalties neurodegeneratives, es poden obtenir cèl·lules precursores (stem cells) de teixits del propi organisme per a la seva re-implantació un cop modificades genèticament Teràpia amb cèl·lules embrionàries ? Clonació cel·lular ? Consideracions ètiques DNA genetically modified cells transgenic or knock-in stem cell