The overall goal of this method is to achieve stable integration of a gene of interest into the human genome of primary cells by the transcriptionally regulated Sleeping Beauty system packaged into integration defective lentiviral vectors. This method can help answer key questions in gene therapy field such as integration of the gene of interest into primary cells using Sleeping Beauty system. The major advantages of this technique is that the inducible hyperactive Sleeping Beauty system is efficiently delivering primary cells resistant to several transfection methods.
Demonstrating the procedure will be Daniela Benati, a post-doc in my lab. Prior to the day of transduction, the human primary keratinocytes are grown as described in the text protocol. To detach subconfluent keratinocytes in culture, remove the medium, wash the cells with 1X PBS, and add one milliliter pre-warmed trypsin working solution to each well.
Incubate at 37 degrees Celsius for 15 minutes in the cell culture incubator. Resuspend the cells of a well thoroughly and transfer the cell suspension to a centrifuge tube containing two milliliters of serum-containing medium. Rinse the well once with three milliliters of 1X PBS and add the rinsed solution to the centrifuge tube with the cell suspension.
Centrifuge at 580 times g for five minutes and discard the supernatant. Wash the cells with five milliliters of 1X PBS, centrifuge at 580 times g for five minutes, and discard the supernatant. In a 15 milliliter polypropylene tube, dilute 1.6 times 10 to the fifth keratinocytes in one milliliter of CFAD medium.
Prepare one dilution for each condition. The three Integration Defective Lentiviral or IDLV vectors were prepared earlier as described in the text protocol. IDLVTKSB carries the TET-regulated hyperactive SB100X transposes.
IDLVrtTA codes for the modulator and IDLVT2 carries the gene of interest. GFP in this example flanked by transposon inverted repeats. Dilute the lentiviral vectors in one milliliter of CFAD medium in the presence of 20 microliters of polybrene as indicated in this table.
One, two, and three are negative controls without vectors. Four and five contain IDLVTKSB and IDLVrtTA. Six, seven, eight, and nine contain all three lentiviral vectors.
Transduce cells in suspension by adding each lentiviral vector dilution to the corresponding keratinocytes suspension. Remove the medium from lethally irradiated 3T3-J2 feeder cells that receded a day ago and plate the transduced keratinocytes onto them. After transducing a well, proceed with the next one.
Incubate at 25 degrees for 30 minutes. Then transfer the cells to 37 degrees for six hours. After six hours, add one milliliter of CFAD medium to each well.
Add one micromolar of doxycycline to wells five, eight, and nine. Return cells to 37 degrees Celsius. Subsequently, process the cells in each well as described in the text protocol.
For cytofluorimetric analysis, the transduced primary keratinocytes detached at day three and day six must be discriminated from the 3T3-J2 feeder layer by labeling the cells with mouse monoclonal APC conjugated antifeeder antibody. Prepare four milliliters of staining buffer for each sample by combining 5%FBS, 500 millimolar EDTA, and 1X PBS. Wash the detached cells with one milliliter of staining buffer.
Centrifuge at 580 times g for five minutes and discard the supernatant. In a tube for flow cytometry acquisition, resuspend five times 10 to the fourth cells in 100 microliters of staining buffer. Add two microliters of antifeeder antibody at a dilution of one to 50 and mix well.
Incubate on ice in the dark for 30 minutes. After 30 minutes, wash with two milliliters of staining buffer. Centrifuge at 580 times g for five minutes.
Discard the supernatant and resuspend in 200 microliters of staining buffer. Use a flow cytometer configured with a blue laser, a red laser, and filters for detection of GFP and APC fluorescence to acquire APC and GFP signals. The GFP positive cell fraction of the APC negative cell population indicates transduced keratinocytes.
To perform real-time PCR, set up reactions in triplicate in a 96-well plate in a final reaction volume of 25 microliters. Each reaction contains one microliter of cDNA, 12.5 microliters of 2X master mix, 1.25 microliters of primers in 20X probe mix, and 10.25 microliters of sterile water. Run real-time PCR according to this PCR program, one cycle at 95 degrees Celsius for 10 minutes then 40 cycles of 95 degrees Celsius for 15 seconds and 60 degrees Celsius for one minute and hold at four degrees Celsius.
Lastly, analyze the real-time PCR data as described in the text protocol. Expression analysis of HeLa cells transduced with increasing doses of transposes in modulator vectors plus or minus doxycycline showed that high doses of both IDLVs were required to strongly induce SB100X expression. Quantitative analysis of SB100X expression on HeLa cells transduced with transposes vector in the presence or absence of the modulator vector revealed an eight-fold activation over the background in the presence of doxycycline.
The transcriptionally regulated Sleeping Beauty system can also be used in primary cells. SB100X expression on keratinocytes transduced with transposes and modulator vectors showed a 15-fold activation over the off state. The transposition experiment performed in keratinocytes transduced with the three IDLV vectors plus or minus doxycycline indicated that 12%of cells treated with doxycycline stably integrated at least one copy of GFP in their genome.
Once mastered, the vital transduction in primary cells can be done in eight hours, cytofluorimetric analysis in two hours, and expression analysis in three hours if performed properly. While attempting this procedure, it's important to remember to optimize vector dose combination used to transduce primary cells and to achieve the higher expression level of the inducible Sleeping Beauty transposes. Following this procedure, other methods such as quantitative PCR and linker-mediated PCR can be performed in order to answer additional questions such as transposon copy number per cell and integration for filing the genome.
After its development, this technique paved the way for the researcher in the field of gene therapy to explore the application of Sleeping Beauty system in engineering clinical relevant cells. After watching the video, you should have good understanding of how to first transduce primary keratinocytes with the IDLV vectors. Second, measure the expression of hyperactive transposes upon doxycycline condition and finally follow transposition by flow cytometry.
Don't forget that working with viral vectors can be extremely hazardous so precautions such as BCL to contain the level and the disposable supply should always be taken with you while performing these procedures.
