DNA constructs in mammalian cells
After successfully cloning a gene, the next step in analysis of its function and/or regulatory mechanisms is typically to introduce normal or mutant versions of the gene into various cell types. (Our inserts represent the “normal”, or wild- type versions of the genes.) We will use Origene’s “TurboFectin 8.0”, a proprietary formulation of positively charged lipids and histones (nuclear proteins) to facilitate the uptake of our cloned genes (plasmids) into cultured human tumor cells. This process, termed “transfection”, is analogous to the transformation of bacterial cells with plasmid DNA that was done a few weeks ago. What the two processes have in common is the relative inefficiency of foreign DNA uptake by the host cells and the need for a ‘facilitator’ of this process. This can take the form of a chemical reagent (usually positively charged) to complex with negatively charged DNA and allow complex formation with the host cells, or physical methods that temporarily disrupt cell membrane integrity (electroporation). Techniques for transfection of mammalian cells are constantly being improved upon, and a reagent and/or process is valuable to scientists if it delivers a relatively high efficiency of transfection with minimal toxicity to the host cells.
Once the plasmid DNA has entered the mammalian cell and made its way to the nucleus, the cellular machinery transcribes the inserted gene sequence into mRNA; upon reaching the cytosol, the mRNA is then translated into protein (provided, of course, that the proper mammalian promoter sequences are present just upstream of the gene in the plasmid). Several factors affect the amount of the protein that is expressed. These include the amount and quality of DNA that is transfected, the promoter that controls gene expression (an inherent feature of the plasmid), and the effect that the expressed protein has on the host cell. It can be difficult to detect in vivo interactions between two proteins at their ‘normal’ expression levels, if these levels are quite low (and beneath our detection limits). Transfection strategies that result in higher than normal expression levels of foreign genes- together with a ‘tag’ of some sort that allows for easy visual detection in recipient cells (i.e., GFP)- can facilitate the study of the transfected protein’s effects. However, results from ‘over-expression’ systems must be interpreted with caution due to the possibility of introducing artifacts (non-physiological events) caused by artificially high protein levels.
In general, when setting up a transfection experiment, a DNA sample that corresponds to a negative control, or “mock transfection” (plasmid vector alone with no insert gene) is included. In our case, the negative vector control (provided by Origene) will be the same plasmid vector that contains your insert, but minus the insert itself. It should therefore still result in expression of the GFP tag in our cultured cells (cytosolic and nuclear GFP), but no ‘fusion protein’. (See sequence file of the vector in Bb.)
In your GFP plasmids, recall that the genes for the fusion proteins are just downstream of strong mammalian promoters (regulatory elements) that result in a constant, high-level of expression in the cultured tumor cells. So, the cells won’t need to be ‘activated’ or stimulated with growth factors or any other kinds of treatments, in order to express the GFP-tagged proteins.
In this ‘transient transfection’, we have a relatively short window of time (24-48 hrs) in which to assess the fusion protein’s effects on cells. We wish to know whether it is expressed in the appropriate cellular compartments (membranes, cytoskeleton, etc.), and also would like to assess a biological effect of the transfected protein on the cell. Our upcoming experiments and procedures will attempt to address these issues.
Since only a fraction of cells will actually take up the plasmid, a functional assay of the entire ‘batch’ of cells may yield a high ‘background’ of untransfected cells that obscures the effects of the fusion protein in cells that were transfected. One type of assay that will avert this problem is a luciferase ‘reporter’ assay of gene transcriptional activity, in which a luciferase ‘reporter’ plasmid is co- transfected with your gene of interest. Upstream of the luciferase gene is a short sequence of DNA (promoter element) that is recognized by the cells’ endogenous transcription factors. Unlike the constant, high level of protein expression that your GFP plasmids will produce, these luciferase plasmids are ‘inducible’: the more transcriptional activation that occurs in cells, the higher the level of luciferase produced and detected. By definition, only the transfected cells will be detected in this type of assay, and therefore the ‘background’ of untransfected cells will not be a problem.
The transcriptional activation pathways under study in our system are some of the pathways that occur during apoptosis induction or the stress/inflammatory responses in cells. These pathways involve the transcription of a variety of genes whose protein products are required to carry out the functions of apoptosis, DNA replication/repair, and/or cytokine secretion. Transcription of a battery of ‘related’ genes is often carried out by a single type of transcription factor complex (eg, called AP-1, p53, SRE, NFAT, or NF-kB, etc.) acting on common promoter sequences in these different genes.
If our cultured tumor cells are induced to activate these signaling pathways, will the presence of our transfected GFP-fusion protein influence the degree of activation? This can be quantitated in our luciferase assays.
In addition to the ‘inducible’ luciferase reporter plasmids (see below), we will also include very small quantities of a ‘control’ luciferase plasmid (Renilla- luciferase) whose expression should remain constant in the cells despite any kind of treatment. This control plasmid will account for differences in cell number or transfection efficiency between wells and treatment conditions, and should help to reduce variability in our duplicate samples.
For this first ‘round’ of transfections, we (SAC and Joanne) will guide the selection of recipient cells and luciferase reporters (and drugs) for each team; once the results are available and there is time to analyze, you will have an opportunity to re-design/optimize/fine-tune the assays for a second round. Also, an opportunity to visualize your transfected cells under fluoresce microscopy will be available during our second round of transfections in glass-bottomed ‘MatTek’ dishes (which will be a much simpler procedure).
Please select one of your two plasmid samples for transfection: looking at all of the data from OD readings and gels, choose what appears to be the ‘better’ of the two.
Transfection consists of two basic steps: (1) preparing a ‘complex’ in serum-free media of all the plasmid DNA (3 different plasmids) plus the transfection reagent (Turbofectin 8 in this case); (2) addition of this complex to the cells; then the cells do the rest!
Referring to the 12-well plate diagram at the end of this document, you can see that 6 wells are allocated to the ‘vector GFP’ control plasmid, and the other 6 are allocated to your gene-GFP. All wells in a single plate will include the same luciferase reporter plasmids. Since you need a minimum of 6 wells’ worth of complex for ‘vector’ samples and another 6 wells’ worth for ‘gene’ samples, we will re-visit the ‘master mix’ concept (from PCR) and simplify the set-up. Unlike PCR master mixes, however, there is no single ‘final volume’ per well that must be maintained, since it is the quantity of DNA (ug or ng) that is most important. Final volumes per well will typically be ~56 ul, give or take a small amount.
The inducible ‘Reporters’:
- AP-1-RE Luciferase: Activator Protein -1 (AP-1) Response Element; AP-1 is a widely acting heterodimeric (2 unequal subunits) transcription factor comprised of the proteins Fos and Jun; it is often found in hyperactivated form in cancer cells. In response to cytokines, growth factors, and other stimuli,
AP-1 regulates gene expression leading ultimately to cell proliferation.
This reporter plasmid (below) contains 6 copies of an AP-1 binding site (response element, RE) that drives transcription of the luciferase reporter gene when activated. A positive control for this reporter is the phorbol ester PMA (Phorbol Myristate Acetate), a potent tumor promoter that activates the signal transduction enzyme Protein Kinase C-á. (PKC-á ultimately activates AP-1 transcription factor activity.)
AP-1 Luciferase reporter plasmid
- p53-RE- Luciferase: p53 Response Element. p53 is transcription factor (protein) that functions as a tumor suppressor. In response to a variety of stressful stimuli, particularly those inducing DNA damage, p53 is activated post-translationally by mechanisms (eg, acetylation) that increase its stability in cells; other covalent modifications (including phosphorylation) alter its conformation and allow it to function as a DNA-binding transcription factor.
p53, by binding to its “response element” (recognition sequences in DNA), regulates the expression of a large variety of genes involved in DNA repair, and genes whose products halt the cell cycle while repair mechanisms are carried out. Alternatively, if the DNA damage is irreparable, p53 will induce expression of genes that bring about apoptosis of cells.
In this reporter plasmid (below), two copies of a p53 binding site (response element, RE) are upstream of the luciferase reporter gene, and these RE’s drive transcription of luciferase when activated (for example by Mitomycin C, which cross-links DNA).
p53-Luciferase reporter plasmid
- NFkB-RE-Luciferase: NF-kB is a transcription factor complex that is present in the cytoplasm, sequestered by an inhibitor, and thus ‘ready to respond’ rapidly when cells encounter harmful stimuli. Activation of this transcription factor complex- by phosphorylation and inactivation of its inhibitor- is downstream of a variety of cellular stimuli (notably, TNFá). This transcription factor regulates genes responsible for the innate and adaptive immune responses, as well as genes that control cell proliferation and survival. This reporter plasmid (below) contains the NF-kB binding site (response element, RE) just upstream of luciferase.
NF-kB Luciferase reporter plasmid
- SRE-luciferase: “Serum Response Element”. Downstream of the activation of MAPK/ERK signaling pathway, transcription factor binding to this DNA response element controls the transcription of many genes including those involved in cell cycle regulation, apoptosis, differentiation.
- NFAT-RE-luciferase: “Nuclear Factor of Activated T cells” Response Element. The transcription factors that bind to this response element are important in immune responses and certain developmental programs. Research of JP has shown that transcription from this ‘response element’ results from RASSF1A overexpression in cultured leukemia cells; in turn, this is believed to result in production and secretion of FAS ligand, an apoptosis- inducing factor. Would RASSF1A overexpression in ‘non-leukemia’ tumor cells also activate transcription at NFAT (and possibly, FAS ligand production)? Or, is its tumor suppressor function through a different mechanism?
Set-up for transfection reactions in 12 well plates
Two different human tumor cell lines (U2OS osteosarcoma and HT-1080 fibrosarcoma cells) will have been prepared for transfection into 12-well plates. On the day before transfection (Mon 10/27), 1 x 105 cells per well will be plated, in order to have ‘subconfluent’ cells that will more readily take up plasmid DNA (as compared to dense, confluent cells).
Vector ‘GFP’ plasmids and luciferase reporter plasmids:
-tGFP vector plasmid = 0.5 ug/ul;
-Renilla-luciferase ‘control’ reporter plasmid = 1 ng/ul
Inducible luciferase reporter plasmids
-p53-luciferase plasmid = 0.6 ug/ul
-AP-1-luciferase plasmid = 0.3 ug/ul
-NF-kB-luciferase plasmid = 0.3 ug/ul
-SRE-luciferase plasmid = 0.5 ug/ul
-NFAT-luciferase plasmid = 0.3 ug/ul
We are using a 500:1 ratio of Firefly (inducible) luciferase: Renilla (control) luciferase, based on previous trials
The following steps are performed inside the tissue culture hood:
- Label two eppendorf tubes, for each Master Mix*: “vector” and “gene ‘x’”; add the required volume of serum free media (SFM) to each tube.
- Add the required volume of Turbofectin 8 (T8) to the tubes; mix by gentle pipetting. Let incubate (sit) for 5 min at room temperature.
- Add the plasmid DNAs as indicated (3 different plasmids!). Mix by gentle pipetting. Let sit for ~20 min at room temperature. During this time, the DNAs and TurboFectin will complex together due to electrostatic interactions.
- Pipette the indicated volumes of complex (see above) into the appropriately labeled wells. (‘Vector’ goes into wells #1,2,5,6,9,10; ‘Gene’ goes into wells #3,4,7,8,11,12) Pipette ‘drop by drop’, rotating around the well. Gently mix the plate side to side to evenly disperse the DNA.
- Cover the dish and place back into the incubator.
After 36 hrs, transfected cells will be treated with appropriate drugs/reagents as we will discuss.
*Example of Master Mix set-up, using GFP ‘vector’ plasmid:
Volume of complex per well x 8 (“master mix”)
- 50 ul SFM 400 ul
- 4 ul T8 32 ul
- 1 ul GFP vector plasmid (0.5 ug) 8 ul
- ‘x’ ul inducible luciferase reporter (0.5 ug) ‘x’ ul
- 1 ul Renilla-Luc (1 ng) 8 ul
From the left column, add up the volumes of everything listed to calculate the total volume of complex to add per well.
After incubation of the master mix (step #3 above), dispense the appropriate amount of complex into each well that will receive vector plasmid (#1,2,5,6,9,10).
Similarly, Master Mixes for your ‘Gene-GFP’ will substitute your plasmid for the ‘GFP vector plasmid’ illustrated in the example above. Luciferase reporter and Renilla luciferase plasmids, of course, will remain the same. ‘Gene-GFP’ master mix will be dispensed into wells #3,4,7,8,11,12.