§ Cationic lipid
Cationic lipid transfection reagents are suitable for transfecting into a wide variety of dividing cell cultures. Commercial examples include: Lipofectamine / L2000, Dharmafect, iFect, and TransIT TKO. Cationic lipids work by forming lipsomal vesicles that house the siRNA payload and bleb their way through the living cell membrane and into the cytoplasm. The efficiency of this process must be determined in order to have confidence in the knockdown effects. There are numerous commercial sources for transfection reagents for good reason; there are numerous cell types and lipsome structure will influence transfection efficiency in the multitude of experimental cell types that exist.
§ Polymeric
Polymeric formulations have been developed and optimized for transfection of shRNA plasmid DNA into the nucleus of cultured eukaryotic cells by vendors such as Open Biosystems. Cationic lipids but not polyethylenimine or polylysine prevent transgene expression when complexes are injected in the nucleus (Pollard et al 1998). Polymers but not cationic lipids promote gene delivery from the cytoplasm to the nucleus and transgene expression in the nucleus is prevented by complexation with cationic lipids but not with cationic polymers.
§ In a six well tissue culture plate, grow cells to a 50-70% confluency in antibiotic-free normal growth medium supplemented with FBS.
NOTE: This protocol is recommended for a well from a 6 well tissue culture plate. Adjust cell and reagent amounts proportionately for wells or dishes of different sizes.
NOTE: Healthy and subconfluent cells are required for successful transfection experiments. It is recommended to ensure cell viability one day prior to transfection.
Prepare the following solutions:
NOTE: The optimal shRNA Plasmid DNA:shRNA Plasmid Transfection Reagent ratio should be determined experimentally beginning with 1 μg of shRNA Plasmid DNA and between 1.0 and 6.0 μl of shRNA Plasmid Transfection Reagent as outlined below. Once the optimal shRNA Plasmid DNA:shRNA Plasmid Transfection Reagent ratio has been identified for a given cell type, the appropriate amount of shRNA Plasmid DNA/shRNA Plasmid Transfection Reagent complex used per well should be tested to determine which amount provides the highest level of transfection efficiency. For example, if the optimal shRNA Plasmid DNA:shRNA Plasmid Transfection Reagent ratio is 1 μg:1 μl, then amounts ranging from 0.5 μg/0.5 μl to 2.0 μg/2.0 μl should be tested.
Solution A: For each transfection, dilute 10 μl of resuspended shRNA Plasmid DNA (i.e. 1 μg shRNA Plasmid DNA) into 90 μl shRNA Plasmid Transfection Medium (serum antibiotic free medium).
Solution B: For each transfection, dilute 1 - 6 μl of shRNA Plasmid Transfection Reagent with enough shRNA Plasmid Transfection Medium to bring final volume to 100 μl.
NOTE: Do not add antibiotics to the shRNA Plasmid Transfection Medium.
NOTE: Optimal results may be achieved by using siliconized microcentrifuge tubes.
NOTE: Although highly efficient in a variety of cell lines, not all shRNA Plasmid Transfection Reagents may be suitable for use with all cell lines.
§ Add the shRNA Plasmid DNA solution (Solution A) directly to the dilute shRNA Plasmid Transfection Reagent (Solution B) using a pipette. Mix gently by pipetting the solution up and down and incubate the mixture 15-45 minutes at room temperature.
§ Wash the cells twice with 2 ml of shRNA Transfection Medium. Aspirate the medium and proceed immediately to the next step. NOTE: Do not use PBS as the residual phosphate may compete with DNA and bind the shRNA Plasmid Transfection Reagent, thereby reducing the transfection efficiency.
NOTE: Do not use PBS as the residual phosphate may compete with DNA and bind the shRNA Plasmid Transfection Reagent, thereby reducing the transfection efficiency. For each transfection, add 0.8 ml shRNA Plasmid Transfection Medium to well.
§ For each transfection, add 0.8 ml shRNA Plasmid Transfection Medium to well.
§ Add the 200 μl shRNA Plasmid DNA/shRNA Plasmid Transfection Reagent Complex (Solution A + Solution B) dropwise to well, covering the entire layer.
§ Gently mix by swirling the plate to ensure that the entire cell layer is immersed in solution.
§ Incubate the cells 5-7 hours at 37° C in a CO2 incubator or under conditions normally used to culture the cells.
NOTE: Longer transfection times may be desirable depending on the cell line.
§ Following incubation, add 1 ml of normal growth medium containing 2 times the normal serum and antibiotics concentration (2x normal growth medium).
§ Incubate the cells for an additional 18-24 hours under conditions normally used to culture the cells.
Aspirate the medium and replace with fresh 1x normal growth medium.
§ Assay the cells using the appropriate protocol 24-72 hours after the addition of fresh medium in the step above.
NOTE: Controls should always be included in shRNA experiments. Control shRNAs are available as 20 μg. Each encode a scrambled shRNA sequence that will not lead to the specific degradation of any known cellular mRNA.
NOTE: For Western blot analysis prepare cell lysate as follows:
NOTE: For RT-PCR analysis isolate RNA using the method described by P. Chomczynski and N. Sacchi (1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159) or a commercially available RNA isolation kit.
§ Target cells are SH-SY5Y human neuroblastoma cells.
§ Complete medium: DMEM F-12 medium with 10% serum and Penicillin/Streptomycicn
§ Vectors are copGFP control Lentiviral Particles (sc-108084)
§ Use 24 well plate. Each well has 1.9 mm2, good for working with 0.5 ml medium.
§ Test 5μg/ml Polybrene
§ Each well contains 4×104 cells. Will use Viral Particles at a quantity of 4×105 infection units. The concentration of provided viral particles was 5000 infections units per μl, so I will try 100 μl. The MOI is 10.
§ Plate target cells in 12 well plate 24 hours prior to viral infection. Each well contains 4×104 cells in 0.5 ml complete medium.
§ Monitor the seeded cells and make sure that the cells are around 50% confluent.
§ Bring Polybrene (sc-134220) to room temperature, and complete medium to 37°C.
§ Prepare a mixture of complete medium with Polybrene at a final concentration of 5 μg/ml.
§ Remove media from plate wells and replace with 1ml of Polybrene/media mixture per well.
§ Put the plate back to the incubator until lentiviral particles were thawed.
§ Thaw lentiviral particles at room temperature and mix gently before use. This takes about 5 minutes.
§ Infect cells by adding the indicated amount of shRNA lentiviral particles to the culture.
§ Swirl the plate gently to mix and incubate overnight.
NOTE: Lentiviral particles were thawed at room temperature. As soon as the vial is thawed, Lentiviral particles were immediately added to the plate to avoid prolonged exposure of the particles to ambient temperature. Did not use ice.
§ Remove the culture medium and replace with 1 ml of complete medium (without Polybrene).
§ Incubate the cells for 2 days.
§ Examine GFP positive cells under microscope. Found that around 80% cells were GFP-positive, the signal was weak.
Day 5 and on Culturing of Cells before Selection
§ Starting from Day 5, change to fresh medium and wait for cells to reach confluency in order to get enough cells for the selection.
Changed medium on day5, day8.
§ Untreated Cells. Untreated cells will provide a reference point for comparing all other samples.
§ Empty construct, containing no shRNA insert; The empty viral particles or DNA are a useful negative control that will not activate the RNAi pathway because it does not contain an shRNA insert. It will allow for observation of cellular effects of the transduction/transfection process. Cells transduced/transfected with the empty control provide a useful reference point for comparing specific knockdown.
§ Non-targeting shRNA; This non-targeting shRNA is a useful negative control that will activate RISC and the RNAi pathway, but does not target any human or mouse genes. The short hairpin sequence cotnains 5 base pair mismatches to any known human or mouse gene. This allows for examination of the effects of shRNA transduction/transfection on gene expression. Cells transduced/transfected with the non-target shRNA will also provide useful reference for interpretation of knockdown.
§ Positive shRNA knockdown control; This control contains shRNA sequence that targets GFP expression. This shRNA control has been experimentally shown to reduce GFP expression. This control serves to quickly visualize knockdown in cells expressing GFP.
§ Positive shRNA knockdown control; This control contains shRNA sequence that targets eGFP expression (GenBank Accession # pEGFP U476561). The shRNA has been experimentally shown to reduce eGFP expression by 90% in C166-GFP mouse fibroblast cells 48 hours post-transduction by mRNA transcript level. This control serves to quickly visualize knockdown in cells expressing eGFP.
§ Positive reporter vector or lentiviral particles; This is a useful positive control for measuring transduction/transfection efficiency and optimizing shRNA delivery. The GFP Control contains a gene encoding GFP, driven by the CMV promoter. This control provides fast visual confirmation of successful transduction/transfection.
The copGFP protein is a novel natural green monomeric green fluorescent protein cloned from copepod Pontellina plumata, a type of plankton. The copGFP protein is a non-toxic, non-aggregating protein with fast protein maturation, high stability at a wide range of pH (pH 4-12), and fluorescent properties that do not require any additional cofactors or substrates.
Due to its exceptional properties, copGFP is an excellent fluorescent marker that can be used instead of EGFP (the widely used Aequrea victoria GFP mutant) for monitoring delivery of lentiviral constructs into cells. The copGFP protein has a very bright fluorescence that exceeds the brightness of EGFP by approximately a third.
The copGFP protein emits green fluorescence with the following characteristics:
§ emmision wavelength max – 502 nm
§ excitation wavelength max – 482 nm
§ quantum yield – 0.6
§ extinction coefficient – 70,000 M-1 cm-1
When assaying cells, DO NOT fix with methanol and minimize exposure to light. PFA/Formalin fixation works.
Factors Influencing Successful Transfection
Concentration and purity of nucleic acids
Determine the concentration of your DNA using 260 nm absorbance. Avoid cytotoxic effects by using pure preparations of nucleic acids.
DNA:In terms of plasmid preparation, McManus Lab has not observed a need to use E. coli cells that are highly defective for recombination. High DNA quality usually means high transfection efficiency. All DNA preparations should be performed by Cesium prep or endotoxin-free ion exchange plasmid purification methods. If poor transfection is consistently observed, it may be worth performing a additional clean-up of the DNA. The transfection protocols described here are sensitive to the amount of DNA. It is important to optimize DNA:Transfection Reagent ratios.
Transfection in serum-free media
The highest transfection efficiencies can be obtained if the cells are exposed to the transfection complexes in serum free conditions followed by the addition of medium containing twice the amount of normal serum to the complex medium 3–5 hrs post transfection (leaving the complexes on the cells). However, the transfection medium can be replaced with normal growth medium if high toxicity is observed.
No antibiotics in transfection medium
The presence of antibiotics can adversely affect the transfection efficiency and lead to increased toxicity levels in some cell types. It is recommended that these additives be initially excluded until optimized conditions are achieved, then these components can be added, and the cells can be monitored for any changes in the transfection results.
High protein expression levels
Some proteins when expressed at high levels can by cytotoxic; this effect can also be cell line specific.
Cell history, density, and passage number
It is very important to use healthy cells that are regularly passaged and in growth phase. The highest transfection efficiencies are achieved if cells are plated the day before. However, adequate time should be allowed to allow the cells to recover from the passaging (generally >12 hours). Plate cells at a consistent density to minimize experimental variation. If transfection efficiencies are low or reduction occurs over time, thawing a new batch of cells or using cells with a lower passage number may improve the results.
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