of HT22 Cells derived from Mouse Hippocampus
Sections I and II
Biology
Section I
1. Introduction
Fluorescent protein development research has gained increased knowledge about the range in the color palette available from the Aequorea Victoria jellyfish. The wavelength emissions in the blue to yellow range are now better understood. [1] Another area that has seen a great improvement in knowledge is the understanding of emissions of the monomeric fluorescent protein emissions in the orange to far-red spectral region. [1] The spectral region of fluorescent in the orange to far-red is derived appropriate reef coral species. [1] This area of research is particularly exciting because of the work from the National High Magnetic Field Laboratory, Florida State University which suggests “that almost any biological parameter can be investigated using the appropriate fluorescent protein-based application.” [1]
Transfection protocol has been developed making it possible to insert nucleic acids into eukaryotic cells. Transfection is an important laboratory procedure that helps researchers better understand the purpose of a protein in a selected cell and identify its properties. Transfection is used for gene transcription activity and factors research as well as other studies on RNA, protein-protein interactions’ and the translation process. Generally for the transfection process DNA in the plasmid form is used. This laboratory is using the chemical method to study transfection because has been shown to be the most effective and requires easily acquired concentrations of cells. “The technical advances in widefield fluorescence and confocal microscopy . . . have demonstrated invaluable service in many thousands of live-cell imaging experiments.” [2] This laboratory has used the transfection methodology with live cells and identified the cells using fluorescence and conical microscopy. The four plasmids that were transfected were the Green Fluorescence Protein (GFP), Methyl CpG-binding protein (MeCP2-GFP), Tau and the Silent mating type Information Regulation 2 homolog 1(SIRT1). The cells used for transfection were HT22 cells derived from the mouse hippocampus. GFP, MeCP2, Tau-Flag and Sirt1-Flag, unknowns, were given to each group to identify.
Description of the two proteins Tau and SIRT1
Tau
The Tau expression plasmid is a protein associated with microtubules and can enhance the stabilization of the microtubules located in the neuronal axons. [3] The highest concentrations of Tau proteins in the body are located in the axion neurons of the central nervous system. [3]The function of stabilization of microtubules is very important because when the tau proteins are not working properly causing destabilization of the microtubules, the disease caused in the human body can be Alzheimer’s and other types of dementia. [3] A single gene, microtubule-associated protein tau (MAPT) when treated by alternative splicing produces Tau; when Alzheimer’s Disease (AD) is present in a brain the tau has become aggregated due to its phosphorylations; this leads to intracellular formalin of fibrillary tangles in neurons present. [3] researched the problem, what could be the physiological substrate fro protein phosphatise (PP) 5. A mammalian brain was used because of the high expression of PP 5. [3] The researchers concluded that “TAU is probably a physiological substrate of PP5 and that the abnormal hyperphosphorylation of tau in AD might result in part from the decreased PP5 activity in the diseased brains.” [3]
SIRT1
Silent mating type information regulation 2 homolog 1 is known as situin 1 and has been shortened to SIRT1. [4] The SIRT1 protein is activated by resveratrol and the result is a longer life for vertebrate systems. [4] Resveratrol is a phenol which is found in large concentrations in red grape skins. SIRT1 is a deacetylase which means it deacetylates proteins with the particular function of protecting cells from stressors (“cellular stress response and energy metabolism”) which enhances longevity. [4] SIRT 1 is associated with protecting cells so they do not die. SIRT 1 is also known as the NAD+-dependent protein deacetylase actor and many studies have tried to learn more about its functions and properties. Guo (et al., 2012) reported that the regulator of the NAD+-dependent protein deacetylase is its oligomeric status (how many sub-units make up the protein). [4] The researchers observed that “nonphosphorylated SIRT1 protein is aggregation-prone . . . conversely phosphorylated SIRT1 protein is largely in the monomeric state and more active.”). [4]
The amount of Tau and SIRT1 is an important area of Alzheimer’s disease research. An example of the type of research being accomplished is the observation of the correlation between the accumulation of tau and the decreased concentrations of SIRT1. [5] The overall purpose of SIRT1 research is to understand how it manages to “increase the lifespan through regulation of cellular metabolism.” [5] Human subjects were used to compare the concentration of SIRT1 (sirtuin1) “in the brains of AD patients and controls using Western immunoblots and in situ hybridization.” [5]
Research Problem
Transfection can be accomplished using three different methodologies: chemistry (using reagents), physical (such as electronic stimulus), and virally (with antibiotics or viruses). The basic idea is to introduce a nucleic acid, in this case DNA, into a one celled sample. Chemical reagents are effective and necessary because nucleic acids and the plasma membrane are negatively charged. The other variables that make the introduction of introducing the nucleic acids into a cell include the nucleic acid size and the lipid membrane hydrophobicity (water repellence). The purpose of the chemical reagents is to neutralize the nucleic acid charge and can even be used to lessen the lipid negative charge. Lipid-based reagents not only aid in neutralizing the lipid charge but also enhance the process by triggering endocytosis or the lipid-based reagent may fuse with the membrane.
2. Materials and Methods
Transfection materials needed
The necessary reagents needed to prepare the reagents included 20 ml PBS; 4 ml Paraformaldehyde (4% w/v in PBS); 40 μl lipofectamine; 4 tubes with DNA; 1 ml OptiMEM; 1 ml Sterile H20; 70% ethanol made in ice cold PBS. The fixing of the transfected cells required the use of DAPi and paraformaldehyde. Paraformaldehyde (PFA) is a hazardous material so safety precautions were taken and was discarded into the PFA container, especially there for the purpose. Step 3, the immunocytochemistry required a blocking serum made of 5% BSA/5% Goat serum in PBS. 0.2% Triton. HT22 cells derived from the mouse hippocampus: the primary antibody (mouse anti-FLAG in a 1:1200 dilutio), and a secondary antibody (Goat anti-Mouse conj. Texas Red, 1:800 dilution), Fluromount, RNAse were needed also.
Pipettes in 3 sizes: P1000, P200, P20 were used. Parafilm and a box top were used to make a grid to hold samples which were covered with coverslips. Sharp-tipped forceps, microscope slides,
Instrumentation
Confocal Microscope to provide the data and a flash drive to save data
Methods: Step 1. Transfection of animal cells
Two of the 1.5 ml tubes containing the plasmids (DNA) were appropriately labeled and 55 μl were added to both of the Tubes A and B. 2 μl of Lipofetamine was added to Tube A. Two μl of plasmid was added to Tube B. Each tube was gently mixed (vortex) and then incubated for 5 minutes at room temperature. The mixture in Tube A was incubated for 20 minutes in dark room at room temperature. After the 20 minute incubation Tube A was ready to be put into the wells. 100 μl of the solution (now mixed and incubated) was placed drop wise into each of the wells. The tray with the filled wells was then set aside until they were fixed five days later.
Methods: Step 2. Fix the Transfected cells
Pre lab preparation: DAPI is prepared as a 1:10,000 dilution in PBS; 70% ethanol with PBS
The medium was aspirated from the cells and then 500 μl of ice-cold PFA. A special method was used to carry out the aspiration of the medium from the wells; the aspiration was carried out slowly in order to avoid liquid ‘jumping’ into pipetman shaft. After the aspiration was completed the tray was incubated on ice for 20 minutes. At this point the plasmids in Tube A were treated differently from the plasmids in Tubes X and Y. The plasmids in Tube A were washed with 500 μl of cold PBS, and then the DAPI is added, then the cells are washed with 500 μl cold PBS. 500 μl 70% ethanol is added to Tubes X and Y.
Methods: Step 3. Immunocytochemistry
Five days after the fixing of the transfected cells the immunochemistry step was carried out for the cells from Tube A, X and Y. First the ethanol needed to carefully aspirated off the cells, and then washed with 500 μl cold PBS. Then 500 μl of 0.2% Triton was added to the tubes which were then incubated for 5 minutes and then aspiration was carried out again to remove the Triton wash. Next 300 μl blocking serum was added and then the cells were incubated in a dark room for 30 minutes at room temperature. During the incubation period parafilm was placed on top of a box top and then flattened. A grid was drawn into the flattened parafilm and each square was labeled to point out the place for the coverslips. After the 30 minute incubation 35 μl drop of primary antibody was added to each square of the prepared grid. The next step had to be done very carefully because the sharp-tipped forceps to place a coverslip into each well with the cell-side down on top of the antibody spot. The blocking solution needed to be aspirated and throw away. Then 500 μl cold PBS was added to each well. Another parafilm grid was made in the same way and the squares were each labeled like the first parafilm grid. 35 μl of the secondary antibody was added to each square. The same forceps were used to place the coverslips appropriately on the antibody spots with the cell-side down. The samples were incubated for 45 minutes in the dark room. The next step after the incubation was to transfer the coverslips iwth the cell-side up to the 4-well tray. 500 μl PBS was added for a wash and then aspirated off. Nest 500 μl nuclear staining solution and 0.5 μl RNAse. The cells in the solution of nuclear staining and RNAse were covered with foil and incubated in ice for 10 minutes. Aspiration was then carried out. The last PBS wash was done with 500 μl PBS. The Fluromount was added 20 μl per slide for each coverslip. Labeling was done. Then the slides were stored in a dark place until two days later.
Methods: Step 4. Conventional Fluorescence and Confocal Microscope
3. Results
The four test tubes – A, B, X, Y- with HT22 cells transfected with plasmids were evaluated with conventional fluorescence and confocal microscope in the last step of the experiment. The transfected efficiency was high indicating that the correct reagents were used and the laboratory was carried out correctly. The tubes labeled X and Y were stained with Syto 13, a nuclear stain. The cellular localization of Tau (the cytoplasmic protein portion of the cell) and SIRT (the nuclear protein part of the cell, DNA) was successful. The SIRT was identified because they were spherical with well defined shape. (See Sect. 1. Fig. 1) The Tau could be identified under the microscope because of the morphology of the cells developing intracellular fiber structures associated with the microtubules. (See Sect.1 Fig. 2)
Tubes A and B held the HT22 cells that were stained with DAPI which is a nuclear stain. The pecent transfection efficiency is calculated as the ratio of the number of cells with green fluorescence over the number of cells which were not transfected. In Tube A approximately 24 BFP are visible after the transfection treatment and two transparent GFP are visible indicating that more cells were not transfixed than were transfixes. So 2/24 x 100 = ~ 8.3%. (See Fig, Section 1 Figure 9 upper left) The two fluorescent GFPs are transparent and the cells have blurry boundaries. For the cells labeled B approximately 4/34 x 10 = ~12 % were transfixed as shown by the amount of fluoresced. GFP (4) compared to the non transfected cells (34). (Section 1 Figure 7 left)
The tau protein is a cytoplasmic protein whereas the SIRT protein is a nuclear protein. The cytoplasm consists of the cytolsol which resembles jello and the organelles. Organelles are all the types of substructures found in the cytoplasm including the eukaryote cells. The cytoplasm is held together by the cell’s membrane. The nuclear protein, DNA, is located in the cell’s nucleus. Tau protein is external from the cell’s nucleus. Tau is associated with the microtubules because tau is necessary to stabilize the microtubules. Both tau and DNA are associated with degenerative diseases like Alzheimer disease and that is why there is a lot of research focused on their function in cells and their possible connection with each other in terms of function. The Tau stabilizes the microtubules and the SIRT is responsible for longevity; therefore they are both types of support mechanisms that have a function of protecting the cell and keeping it alive.
X = SIRT1
Section 1 Figure 1. SIRT1
Y = Tau
Section 1 Figure 3. Tau
Section 1 Figure 9. A Before (on the bottom) and after (on the top)
Section 1 Figure 10. B -
before transfection (right) and after transfection (left).
4. Discussion
The steps preparing the samples were successfully carried out so they could be examined under the microscope. (a) fixation, (b) permeabilization (so fixed cells can penetrate the antibodies), blocking sites that could offer only nonspecific interactions, labeling of the fixed cells so identification and mounting the samples. The use of fluorescence is based on the wavelength and the degree of brightness from that wavelength which can be used for comparison purposes. The laboratory was a success because the transfection did result in some of the HT22 mouse cells becoming transfected using the chemical reagent methodology. The SIRT1 proteins before transfection were shaped as well defined spheres. The tau proteins after tranfection were extremely different in morphology than before transfection. The tau proteins displayed a fiberous growth that was interconnected. The Transfection Efficiency was low; in the control (B) it was ~12% and in the sample (Tube A) it was approximately 8%. The percent transmission efficiency does indicate that the laboratory was carried out in a consistent manner for both the control and the sample because 12% and 8% are close in value.
5. References
[1] Olenych, S.G., Claxton, N.S., Ottenberg, G.K. and Davidson, M.W. (n.d.)The Fluorescent Protein Color Palett. National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida. http://www.microscopyu.com/pdfs/FPColorPalette.pdf
[2] Piston, D.W., Patterson, Lippencott-Schwartz, J. Claxton, N.S. and Davidson, M.W. (n.d.) Introduction to Fluorescent Proteins. Nikon Microscopy, http://www.microscopyu.com/articles/livecellimaging/fpintro.html
[3] Liu, F., Iqbal, K., Grundke-Iqbal, Rossie, S. and Gong, C-X. Dephosphorylation of Tauby Protein Photophatase 5, J. of Biological Chemistry, 280(3): 1790-1796, 2005. http://www.jbc.org/content/280/3/1790.full.pdf+html
[4] Guo, X., Desimer, M., Tolun, G., Zheng, X., Xu, Q, Lu, J. Sheehan, J.K., Griffith, J.D., and Li, X. the NAD+-dependent protein deacetylase activity of SIRT1 is regulated by its oligomeric status. Nature, Scientific Reports 2, Article Number 640, 7 Sept. 2012. http://www.nature.com/srep/2012/120907/srep00640/full/srep00640.html
[5] Julien, C., Tremblay, C., Emond, V., Lebbadi, M., Salem, Jr., N., Bennett, D.A., and Calon, F. SIRT1 decrease parallels the accumulation of tau in Alzheimer Disease. J. Neuropathol. Exp. Neurol. Available at PMC on 2010 January 29. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2813570/
Section II
Figure 1. GFP before transcription (GRFP) Two cells are fluorescing very
clearly while the rest of the sample shows large cells that exhibit no brightness.
Figure 2. GFP after transfection before fixing. The number of GFPs has increased, the cell sizes are smaller, the majority of cells are also brighter. Two GFPs are highly fluorescent.
Figure 3. BFP after transfection The lower left quadrant displays a few more than 120 cells. The right lower quadrant is more crowded with about 150 cells so the slide displays over 400 BFPs. On the other hand about 9 GFPs are visible. Not all of the BFPs are bright, most of them are dull, a medium number are bright and a few are display a noticeable higher brightness than the others. A higher magnification shows the green smudges above and among the BFP do have a defined shape. 20X April 25
Figure 4. Close up of BFP after transfection Note the light greenish smudges that seem to be floating on top of the blue fluorescent protein. The out-of focus green smudges are green fluorescent proteins (GFP). 20X magnification 20 x April 25 Fluorescent Microscopy of histidine (Y66H) produces a blue fluorescent protein (BFP) and examples look similar to this slide. (Olenych et al., n.d.)
Figure 5. RFPApril 25 Y1 Series 006 at a high magnification.
Figure 6. RFP after transfection. (April 25 Y1) The tube is located in the upper right quadrant of the slide. It is a rectangular shape with rough sides (in other words the 4 sides of the rectangle are not smooth.) Fluorescent Microscopy DsRed protein interacts with two adjacent neighbors, one through a hydrophobic interface and the other through a hydrophilic interface resulting in a microtubule can be seen. (Olenych et al., n.d.)
Figure 7. RFP close up microtuble on topA higher magnification and the tubular fusion with the protein is much easier to discern in center of the slide. April 25Y1 April 25, Y1, Series 4
Appenix
Appendix Figure 2. Mutation enhanced GFP
Source: Olenych, S.G., Claxton, N.S., Ottenberg, G.K. and Davidson, M.W. (n.d.)The Fluorescent Protein Color Palett. National High Magnetic Field Laboratory, Florida State University, p. 2.
Appendix Figure 3, Examples of Fluroescent Protein Chimeras
Source: Olenych, S.G., Claxton, N.S., Ottenberg, G.K. and Davidson, M.W. (n.d.)The Fluorescent Protein Color Palett. National High Magnetic Field Laboratory, Florida State University, p. 10
Appendix Table 1. Properties of fluorescent protein variants
Source: Olenych, S.G., Claxton, N.S., Ottenberg, G.K. and Davidson, M.W. (n.d.)
The Fluorescent Protein Color Palett. National High Magnetic Field Laboratory,
Florida State University, page 17.
Appendix Figure 4. Before and after transfection
Source: Lab Handout
