Transfection of Melanoma Cells with Lipofectamine

By Julia Davis

Purpose

• Introduce nucleic acids into eukaryotic cells
• Our DNA: GFP & Cas9
• Big picture
• Currently: melanoma cells
• Next: Calu-3 cells (lung epithelial cells)
• Future: human bronchial epithelial cells
• Cas9 will cleave mutated CFTR gene
• A functional CFTR gene will replace the mutated one

Comparison

• Transfection
• Eukaryotic cells
• Melanoma cells do not have their own mechanism to uptake plasmid DNA from the environment
• Transformation
• Prokaryotic cells
• Bacterial cells easily uptake plasmid DNA from their environment
• Horizontal gene transfer

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Liposome Transfection

• Lipofectamine reagent
• Uses a lipid to allow nucleic acids to cross the cell membrane
• DNA alone cannot easily pass through a lipid membrane
• Packaging DNA in an liposome allows it to cross the membrane and be endocytosed

Methods

Pre-Transfection Plating

• Similar to passaging
• Discard media and add trypsin to decrease cell adherence
• Use hemocytometer to estimate number of cells per µL
• Goal: ~100,000-150,000 cells per well
• Add cells and media to Falcon tube, vortex
• Add cell-media mixture to wells
• One well labeled 0.75, one well labeled 1.5
• Incubate overnight

Methods

Transfection

1. Check for 70% confluency
2. Label 3 eppendorf tubes: 0.75, 1.5, DNA
3. Dilute Lipofectamine 3000 reagent in 2 tubes, creating different final concentrations
1. Add 25µL serum free media to 0.75 and 1.5 tubes
2. Add 0.75µL lipofectamine 3000 to 0.75 tube, 1.5µL to 1.5 tube, flick to mix well

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Methods

Transfection

4. Prepare DNA master mix

• Add 50µL serum free media to DNA tube
• Add 1µg of DNA to DNA tube
• Add 2µL of P3000 to DNA tube
• Flick tube to mix well

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Methods

Transfection

5. Add 25µL of the DNA master mix to each of the dilute

Lipofectamine 3000 tubes, 0.75 and 1.5

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6. Let the mixtures incubate for 10-15 minutes

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Methods

Transfection

7. Add 50 µL from the 0.75 and 1.5 tubes to their

respective wells.

a. When adding the DNA, pipet slowly and in a circle

to mix the DNA into the media.

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8. Incubate cells and analyze

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Results

It worked!

About 2% transfection rate based on Tina and Julia’s estimations

This picture is from Gabe’s transfection

Future

• Mock transfection
• Test our sterile technique
• Don’t want to contaminate other labs
• Clean up Cas9 DNA
• Didn’t work well for Gabe’s transfection
• I only used GFP
• Phenol chloroform extraction
• Use Calu-3 lung epithelial cells instead of melanoma cells
• Secure funding