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Andreia FERREIRA Supervised by Kamilla MALINOWSKA, Didier PICARD Science III, Department of Cell Biology Visualizing gene expression and function at the cellular level Introduction The work allowed me to investigate the profile of protein and DNA expression. The expression of protein can be detected by Western blot or by immunofluorescence using appropriate antibodies. And luciferase assay enables understanding the regulation of protein expression. On the other hand, PCR represents an important method to study DNA. These basic techniques are applied to study the biological processes at molecular levels. In all experiments we studied estrogen receptor α (ERα) that is a nuclear receptor activated by sex hormone estradiol (E2). We investigated also the expression of Hsp90, a chaperone protein of ERα. It stabilizes ERα and protects it from degradation. Western Blot Western blotting is a method to detect proteins with specific antibodies. Proteins are separated according to their size by gel electrophoresis. The blot is a membrane, almost always made of nitrocellulose or PVDF (polyvinylidene fluoride). The gel is placed next to the membrane and application of an electrical current induces the proteins in the gel to move to the membrane where they adhere. The membrane is then a replica of the gel protein pattern, and is subsequently stained with an antibody. Expression of ERα in mammalian (HEK 293T) cells HEK 293T cells were seeded into 6-well plate and transfected with plasmids carrying ERα gene. We had four samples, two with non-transfected cells, and two with the transfected cells. After 48h cells were harvested and lysed with RIPA buffer. This solubilizes the proteins so they can migrate individually through a separating gel. 1 Andreia FERREIRA Supervised by Kamilla MALINOWSKA, Didier PICARD Science III, Department of Cell Biology We added protease inhibitor cocktail to RIPA buffer to protect proteins from proteases that are enzymes that break the peptide bonds that link amino acids together in the polypeptide chain forming the protein. The cells were incubated in RIPA buffer and they were put in the sonnicator to help for the deployment/release of the proteins. The samples were loaded on polyacrylamide gel. We used 10% of gel concentration because studied proteins had low molecular weight. After gel separation proteins were transferred to nitrocellulose membrane. Results The expression of ERα was detected by ERα antibodies in Hek293T cells transfected with ERα carrying plasmid. The expression of ERα was not observed in non-transfected Hek293T 75KD 55KD ER α pl as mi d N on tr an sf ec te d ce lls N on tr an sf ec te d ce lls ER α pl as mi d cells. Conclusions ERα protein was expressed in HEK 293T cells. Immunofluorescence Immunofluorescence technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows visualisation of the distribution of the target molecule through the sample. Translocation of ERα into nucleus after E2 treatment HeLa cells were seeded into 12-well plate and transfected with GFP-ERα and Flag-ERα carrying plasmids. After 24h cells were treated with 100nM E2 for 90 minutes. We had four samples: one with GFP-ERα without E2 treatment One with GFP-ERα with E2 treatment One with Flag-ERα without E2 treatment 2 Andreia FERREIRA Supervised by Kamilla MALINOWSKA, Didier PICARD Science III, Department of Cell Biology One with Flag-ERα with E2 treatment Flag-ERα transfected HeLa cells: the cells were incubated with anti-Flag antibodies and expression was detected by fluorescent microscope. GFP-ERα transfected HeLa cells: expression was detected by fluorescent microscope. Results The expression of Flag-ERα and GFP-ERα was observed in transfected HeLa cells. In samples treated with E2 ERα GFP-ERα non-induced GFP-ERα induced (E2) was detected in nucleus. Conclusion Flag-ERα non-induced Flag-ERα induced (E2) ERα accumulates in nucleus after E2 treatment. Luciferase assay The Luciferase Assay System is an extremely sensitive assay for quantitation of gene expression. Luciferase gene is controlled by regulatory element of our gene of interest. The luciferase reagent is added to cells expressing luciferase enzyme. The processed reagent emits the light that could be detected. Regulation of ERα-target gene expression HEK 293T cells were seeded and transfected with three plasmids: o Plasmid with firefly luciferase gene controlled by estrogen response elements (ERE) 3 Andreia FERREIRA Supervised by Kamilla MALINOWSKA, Didier PICARD Science III, Department of Cell Biology o Plasmid with renilla luciferase gene for normalization o ERα gene The cells were treated with 100nM E2 After 24h, the luciferase activity was measured. Conclusion The expression of firefly luciferase was activated after E2 treatment. Genotyping Genotyping is the process of determining differences in the genetic make-up (genotype) of an individual by examining the individual's DNA sequence. It reveals the alleles an individual has inherited from their parents. Genetic mouse model for ERα chaperone protein Hsp90 Ear samples from mice were collected and PCR that is used to amplify a particular DNA sequence (Hsp90) was performed PCR procedure: o Denaturation: It separates the two stalks of the DNA from each other o Annealing: Taq polymerase (enzyme) catalyses DNA synthesis o Elongation: transcribed RNA is getting elongated The PCR samples were loaded on agarose gel. Results Mice n°1645 and n°1647 expressed Hsp90 from only one allele. Conclusion We found mice model with Hsp90 expression from one allele. 4 Andreia FERREIRA Supervised by Kamilla MALINOWSKA, Didier PICARD Science III, Department of Cell Biology Discussion Molecular biology methods allow getting genetically modified cells by overexpression of genes from plasmid. Then the overexpressed proteins can be studied: Their expression (here ERα expression) can be detected (western blot, immunofluorescence), their activity can also be tested and their effect on target genes (like ERα and luciferase gene with ERE) can be studied. We can also observe that after ligand treatment proteins can translocate like ERα after E2 to nucleus. People work not only with cells but also with living organisms like mice (genotyping). Thanks After a wonderful week in Cell Biology laboratories, I would like to thank Professor Didier Picard and Kamilla Malinowska for all assistance and help, but also for all the time spent with me stirring up my interest. In addition, I would like to thank “La Science appelle les jeunes” for giving me the opportunity of spending an interesting week in a laboratory. For me, it’s memorable and I look forward to my future studies around this domain. 5