Transduction to Label PDX Tumor Cells: Introducing Lentivirus Expressing Fluorescent Marker into the Tumor Cell In Vitro
成績單
Lentivirus is a Retrovirus that contains RNA genetic material. This virus provides an efficient and stable method of introducing transgenes to cancer cell lines. Start by adding neuraminidase to a tube containing Lentivirus that expresses green fluorescent protein, or GFP. Keep the tube at 37 degrees Celsius for one hour to increase the virus's binding efficiency.
Next, centrifuge a tube containing tumor cell suspension to form a pellet. Add mammosphere media containing polybrene to resuspend the cells. Polybrene is a cationic polymer that increases Lentiviral infection efficiency. Now plate the optimal number of tumor cells per well in a six well tissue culture plate. Add the required amount of Lentivirus into each well of the culture plate.
Swirl the plate to mix the content, and keep at 37 degrees Celsius for 96 hours with a continuous supply of carbon dioxide. The Lentivirus enters the cell and releases RNA. The viral reverse transcriptase converts the RNA into double stranded DNA which integrates into the host genome. Place the plate under a fluorescent microscope and monitor cells for successful transduction. The transduced cells fluoresce due to GFP expression.
In the example protocol, we will perform transduction of PDX-dissociated tumor cells with Lentiviral vectors.
To transduce the tumor cells, centrifuge the isolated cells for resuspension in two milliliters of mammosphere medium. After counting, centrifuge the cells again and resuspend the pellet in two milliliters of mammosphere medium, supplemented with 20 microliters of polybrene. Next, plate 2 times 10 to the fifth viable tumor cells per well in an ultra low adherence six well tissue culture plate and Lentiviral particles to the cells at a multiplicity of infection of 10.
Swirl the plate to mix the virus with the cells and incubate the co-cultures at 37 degrees Celsius and 5% carbon dioxide for up to 96 hours, adding 500 microliters of fresh mammosphere medium after the first 24 hours. When at least 10% of the cells are GFP positive, transfer the transduced cells from at least one well into a 15 milliliter conical tube on ice and wash the well with one milliliter of mammosphere medium.
Pool the wash with the cells and centrifuge the tumor cells in 10 milliliters of HBSS plus HEPES. Finally, resuspend the cells in 50 microliters of basement matrix extract on ice and load the embedded cells into a 0.5 milliliter insulin syringe for reimplantation.
