## Fusion Proteins for Transfecting Mammalian Cells: Week 2
**Transfection** is the process of deliberately introducing purified RNA/DNA into eukaryotic cells. While a number of transfection reagents are commercially available nowadays, a quick, simple, efficient and inexpensive method is to transfect eukaryotic cells via calcium phosphate co-precipitation with DNA (Graham and van der Eb, 1973). The insoluble calcium phosphate precipitate with the attached DNA adheres to the cell surface and is brought into the cells by either phagocytosis or endocytosis (Figure 1). Calcium phosphate transfection has been optimized and widely used with many adherent and nonadherent cell lines (Jordan et al., 1996). Calcium phosphate transfection can result in transient expression of the delivered DNA in the target cell, or establishment of stable cell lines (the latter requires a drug selection process). The efficiency of transfection can be close to 100% depending on the cell lines used.
##### Figure 1. A schematic representation of cell transfection by Calcium Phosphate.
### Objectives of the lab:
This week you transfecting mammalian (HEK293T) cells with the plasmid DNA that you isolated from bacteria in last week’s lab. Upon completion of this lab, you should be able to:
1) Understand the mechanism that allows cells to take up plasmid DNA during Calcium Phosphate Transfections.
2) Be able to transfect mammalian cells using the Calcium phosphate method.
## How it works
### Calcium Phosphate Precipitation
The Calcium Phosphate Transfection method for introducing DNA into mammalian cells is based on forming a calcium phosphate-DNA precipitate. Negatively charged (plasmid) DNA is introduced to and interacts with the cationic molecules in a solution of calcium phosphate. The resulting insoluble Calcium phosphate precipitate facilitates the binding of the DNA to the cell surface. DNA then enters the cell by either phagocytosis or endocytosis. Upon entering the cell, the DNA escapes its endocytic compartment. Endocytic escape is not well understood; however, the most accepted hypothesis is that influx of Ca2+ generated by the calcium phosphate-DNA precipitate present in endosomes promotes the influx of water into these compartments resulting in an osmotic rupture of the endosomes and release of the plasmid DNA (this has hypothesis has been called the “proton-sponge” hypothesis (JP Behr, 1997)). The Calcium phosphate method for transfecting mammalian cells is generally very efficient and has been optimized for a variety of cell types including the HEK293T cells you will be transfecting in lab today.
## Procedures
### Generating your DNA-Calcium phosphate precipitates
Working in the same groups as last week, begin by claiming and thawing your plasmid DNA.
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Record your estimated DNA concentrations (determined in last week’s lab) here:
**ER-RFP DNA conc. ___________________ ng/μl**
**mEmerald-ELP-1-25 DNA conc. ___________________ ng/μl**
For your Calcium phosphate transfection, you will need **1 μg** of plasmid DNA for each of your plasmids.
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### Calculate the volume of DNA you will need for your Calcium phosphate transfection:
**Volume of ER-RFP DNA needed (μl): ___________________**
**Volume of mEmerald-ELP-1-25 DNA needed (μl): ___________________**
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## Making the DNA-Calcium Phosphate precipitate:
To make the DNA-Calcium Phosphate precipitates you will need to prepare two solutions: **the DNA-Calcium solution in Tube “a”** and **the Phosphate buffer solution in Tube “b”**. These two solutions will be mixed together to form the DNA-Calcium Phosphate precipitate you will add to your cells.
*BE SURE TO LABEL YOUR TUBES!*
### TUBE “a”
1. Use the appropriate pipette to add 1 μg of ER-RFP plasmid DNA **and** 1 μg of mEmerald-ELP-1-25 plasmid DNA to a new microcentrifuge tube*
2. Add 5μL of 2.5M CaCl2 your microcentrifuge tube containing your DNA
3. Add enough H2O to your microcentrifuge tube to bring the TOTAL VOLUME in your tube to 100 μL.
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### Calculate the volume of H2O you will need to add to TUBE “a” below:
**H2O ___________________ μL**
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### TUBE “b”
1. Use the appropriate pipette to add 100 μL of 2x HBS (Hepes Buffered Saline) to a new microcentrifuge tube*
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To finish making your DNA-Calcium phosphate precipitates you will need to combine the contents of tube “b” with the contents of tube “a”. The precipitate will only form when these solutions are properly combined (Figure 2).
1. Use a p200 pipette to suck up all 100 μL of your solution in Tube “a”
2. Add the solution from Tube “a” dropwise into Tube “b” one drop at a time while gently vortexing the solution in Tube “b”
3. Once you have added all of the solution from Tube “a” into Tube “b” you should see that a precipitate has formed in the tube.
4. Place your Tube “b” into a tube rack to incubate for 30 minutes at room temperature.
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##### Figure 2. Making the DNA-Calcium phosphate precipitate.
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### Transfecting your HEK293T cells
This week you will, once again, be working with the mammalian HEK293T cell line. As the cells must be at the appropriate density (30-90% confluent) for the calcium phosphate transfections to work; this week your cells have already been counted, diluted and plated for you. Each group will receive a 35mm, coverslip bottom dish of HEK293T cells. Once again, as the cells will be plated into a 35mm dish and grown for several days, it will be important to work with the cells in the dish using sterile techniques. A hood with a lot of air flow will once again be used to help prevent contamination of your cells and you should practice sterile techniques when working with your cell culture.
1) Turn the blower on in the cell culture hood (and the light if needed). Wipe down the counter surface of the hood with 70% ethanol.
2) Carefully pour the culture media from your 35mm, coverslip bottom dishes into the liquid waste container containing bleach.
3) With a 2 mL sterile serological pipette, add 1mL of DMEM+ 10% FBS to each of your 35mm, coverslip bottom dish of cells
4) Using a pipette, add the DNA-Calcium phosphate precipitate to your cells
5) Gently tilt the dish side to side to coat the cells with your DNA-Calcium phosphate precipitate
6) 6) Label one dish “CONTROL” and one dish “EXPERIMENTAL”
7) Be sure your lid is on the dish properly and has been well labeled (Date and Initials) and carefully place your plate in the 37°C CO2 incubator in the cell culture room.
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EACH GROUP WILL NEED TO COME BACK AND CHANGE THE MEDIA ON THEIR CELLS 2X & ONE FINAL TIME TO FIX THE CELLS (3X TOTAL).
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### AFTER ~12-16 hours (in the morning).
1) Turn the blower on in the cell culture hood (and the light if needed). Wipe down the counter surface of the hood with 70% ethanol.
2) Carefully pour the culture media from your 35mm, coverslip bottom dish into the liquid waste container containing bleach.
3) With a 2 mL sterile serological pipette, add 1mL of DMEM+ 10% FBS to each of your 35mm, coverslip bottom dishes of cells
4) Be sure your lid is on the dish properly and carefully place your plate in the 37°C CO2 incubator in the cell culture room.
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### AFTER ~48 hours (Wednesday lab--> Friday; Thursday lab--> Saturday).
1) Turn the blower on in the cell culture hood (and the light if needed). Wipe down the counter surface of the hood with 70% ethanol.
2) Carefully pour the culture media from your 35mm, coverslip bottom dish into the liquid waste container containing bleach.
3) With a 2 mL sterile serological pipette, add 1mL of DMEM+ 10% FBS + DMSO to the “CONTROL” DISH
4) With a 2 mL sterile serological pipette, add 1mL of DMEM+ 10% FBS + Brefeldin A to the “EXPERIMENTAL” DISH
## AFTER ~72 hours (Wednesday lab--> Saturday; Thursday lab--> Sunday).
### Fixing your cells:
Each group will need to return to the lab once more approximately 72hours after transfecting their cells to fix their cells (Wednesday lab--> Saturday; Thursday lab--> Sunday). Fixation preserves cells and subcellular structures for later analysis. You will be fixing cells using an organic solvent; methanol. Methanol fixation preserves cells/tissues by dehydration, removing lipids and denaturing and precipitating the proteins in your cells. Moreover, methanol fixation is known to preserve the organization of microtubules in cells making it an ideal fixation method for your experiments.
1) Be sure your lids are on the dishes properly and carefully walk your dishes to the Cell Biology lab.
2) Remove the culture media from your 35mm dishes using a p1000 Pipette tip(liquid and solid waste containers are provided at each bench for proper disposal).
3) Add 1mL of ICE COLD 100% Methanol (Cell Biology Lab freezer) to each of your dishes using a p1000 Pipette tip being careful not to disturb the cells on the coverslips.
4) Incubate the cells in 100% Methanol at -20°C for 20 minutes by placing your dishes in the Cell Biology Lab freezer.
5) Carefully remove the 100% Methanol from your cells using a p1000 Pipette tip.
6) Add 1mL of PBS to each of your dishes using a p1000 Pipette tip being careful not to disturb the cells on the coverslips.
7) Be sure your lid is on the plate properly and carefully place your plate in the 4°C Fridge in the cell biology lab.
## Answer the following questions:
1. What effect does Brefeldin A treatment have on cells?
2. What effect, if any, do you think Brefeldin A treatment will have on the distribution of your ER-RFP and mEmerald-ELP-1-25 proteins? (State your hypothesis.)
3. Based on your hypothesis, state your predictions for the effect of Brefeldin A on the distribution of your ER-RFP and mEmerald-ELP-1-25 proteins in HEK293T cells.
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