A major goal of our team this year has been to find successful ways of increasing mammalian representation within iGEM. The survey we conducted revealed that a lack of resources and easily accessible information has been a major obstacle for other teams. We hope that our detailed and easy to follow protocols will ease the way for future teams hoping to enter the field.
All protocols can also be found on our teaching lab space website to increase accessibility and promote use:
Fluorescent imaging and Flow Cytometry of Adherent Mammalian cell lines
Nucleic Acid Extraction, reverse transcription, and qPCR protocols
Experiments
Each of the following experiments were conducted by our team to optimize transfection efficiency into three mammalian cell lines and further characterize the LACE system using that information. The protocols above were used.
Each test was conducted in a 24-well plate with randomized positions to limit possible edge effects and included a cells only control, providing a baseline that allowed us to accurately gate our flow cytometry data. All expression levels recorded from imaging were calculated as #florescent cells/#total cells (viewed in bright field) per image and extractions of the optogenetic system were done in a dark room with red light only so as to avoid accidental system activation during handling.
Experimental Overview:
Optimizing Transfection Efficiency
Preliminary seeding test
DNA concentration tests
Lipofectamine Concentration Tests
Suspended vs Adhered Transfections
Lipofectamine3000 Transfection vs Calcium Phosphate Transfection
Wash vs no wash
LACE System Activation
Plasmid ratios using multi-plasmid fluorescent system & Plasmid Dye
Single vs Multiple sgRNA test for targeting efficiency
LACE Activator Strength Comparison & NEU activator strength
Light Intensity Comparison
Leakiness Test
1. Seeding Density
The final surface area of each cell line differed greatly once adhered and can further vary based on transfection-related stress. We aimed to experimentally determine the optimal seeding density of each of our three cell lines (NIH-3T3, CHO-DG44, and AML-12) in a 24 well plate prior to beginning transfection.
2. DNA Mass & Concentration
According to ThermoFisher's Lipofectamine3000 protocol, they recommend using 500ng of DNA for each transfection, we wanted to verify this number experimentally. We tested a range of DNA masses on either side of this recommended value but our mini- and midi-preps generally produced lower concentrations. To see if use of a speedvac was necessary to increase the concentration of the plasmid stocks, we tested different concentrations to determine if the dilution factor influenced transfection efficiency.
3. Lipofectamine 3000 Volume
There were two different recommended Lipofectamine3000 volumes according to the ThermoFisher protocol, and it advised testing both for your experimental conditions so we experimented within all three cell lines. For the 24-well plate volumes, they used 1.5ul and 0.75ul Lipofectamine3000.
4. Suspended vs Adhered Transfection
When cells adhere to the surface of the well, occasionally layering on top of one another, they flatten out and limit the membrane surface area exposed to the transfection solution. We wanted to determine if it was better to transfect the cells directly after seeding, ensuring cells are in suspension and have maximized surface area exposed, or if giving them 12hrs to adhere to the plate before transfecting leads to higher efficiency.
5. Lipofectamine vs Calcium Phosphate Transfection
Multiple modes of chemical transfection exist and can be adapted for improved use within specific contexts of experimental conditions. While we had begun with Lipofectamine3000 transfections, we were given access to reagents for a Calcium phosphate transfection and wanted to conduct a cross comparison. The Calcium Phosphate protocol can be foundhere and the adapted ThermoFisher Lipofectamine 3000 transfection protocol can be found in the protocols above.
6. Washed vs Unwashed Transfection
Prior tests (see seeding density test above) have shown that wells transfected with plasmid and Lipofectamine have a significantly lower final cell count than untransfected control wells. An optional "wash step" of aspirating media containing lipofectamine from transfected wells, followed by a rinse with fresh media, then a refill of new media conducted 2 hrs post-transfection may limit the toxic effects of the lipofectamine3000 and increase transfection efficiency.
LACE SYSTEM ACTIVATION
1. Plasmid Ratios
We have already experimentally determined the optimal amount (measured by mass) of DNA for a single plasmid transfection into mammalian cells (see experiment 2 above) but making that a multi-plasmid system increases the difficulty of the transfection. Using our three plasmid GFP LACE testing system, we set out to determine what the optimal ratios of the three plasmids would be to maximize system expression and transfection efficiency measured by GFP expression.
2. Number of sgRNAs in LACE Targeting System
Previous literature (Chavez et al. 2015) observed that increasing the number of guide RNAs in the LACE targeting system improved system ability to bind the target gene. We tested this hypothesis and also validated the quality of each of our designed sgRNA parts in a side by side comparison of activation levels.
3. Activator Strength & NEU Comparison
Different activators attached to the dimerizing proteins of the LACE system have the potential to activate the endogenous target gene to different levels. We have compared the most common activator (VP64) to a new activator that has never been used with the LACE system before but has been shown to cause higher fold increase in other CRISPR-based systems, VPR. Additionally, we compare both of these values to the fold increase of the VP64 activator used by NEU China 2016 iGEM team.
4. Light Intensity
Past studies of the LACE system discussed a wide range of different intensities and settings of light exposure for system activation, frequently using different units and conditions which made cross-study comparison difficult. In addition to temperature tests conducted in the light box to determine a healthy light intensity range for cell growth (see Hardwarefor more information), we also experimentally determined these conditions using system activation.
5. System Leakiness
By measuring mRNA fold increase over cells only of the transfected LACE system in the dark, we can determine how leaky the system is. If the system is completely inducible (not-leaky) then there will be 0 fold increase.
