TITLE: Generation of stable cell lines via retroviral or lentiviral transduction
AUTHORS: Rotimi Fasimoye, Dario R Alessi

[DESCRIPTION]
The ability to stably express a protein of interest in cells is critical to study its function. Here, we describe a protocol to generate stable cell lines using a retrovirus system that can be used for a variety of mouse and human cell lines. Our protocol includes the production of retroviruses encoding the transgene of interest in HEK293FT and the subsequent transduction of the cell line that is intended to stably express the protein of interest. The same protocol can also be used to generate stable cell lines using a lentivirus system. It should be noted that when using this method, the transgene of interest will be randomly integrated into the cell genome.

[GUIDELINES]
References:

Bozena Szulc, Paulina Sosicka, Dorota Maszczak-Seneczko, Edyta Skurska, Auhen Shauchuk, Teresa Olczak, Hudson H. Freeze and Mariusz Olczak (2020). Biosynthesis of GlcNAc-rich N- and O-glycans in the Golgi apparatus does not require the nucleotide sugar transporter SLC35A3. Journal of Biological Chemistry 295 (48), 16445-63. ISSN 0021-9258. https://doi.org/10.1074/jbc.RA119.012362.

[STEPS]
SECTION: Packaging SLC35A2-2xFLAG plasmid into a Retrovirus system
1. 
 
Grow HEK293FT cells to 50-60% confluency in Growth media in a   Petri Dish.
SECTION: Packaging SLC35A2-2xFLAG plasmid into a Retrovirus system
2. Prepare a transfection mix in a sterile 1.5 ml Eppendorf tube, containing:

3.8 µg pGag/Pol plasmid
2.2 µg pVSVG plasmid
6 µg pBABE-SLC35A2-2xFLAG plasmid
300 µL OptiMem

SECTION: Packaging SLC35A2-2xFLAG plasmid into a Retrovirus system
3. Prepare PEI mixture in a sterile 1.5 ml Eppendorf tube, containing:

20 µL 1 mg/mL PEI Max 40K dissolved in distilled water.
300 µL OptiMem.
SECTION: Packaging SLC35A2-2xFLAG plasmid into a Retrovirus system
4. Incubate each mixture (from steps 2 and 3) separately for ~5 min at Room temperature, then combine.
SECTION: Packaging SLC35A2-2xFLAG plasmid into a Retrovirus system
5. Mix by vortexing and incubate at Room temperature for 30 min.
SECTION: Packaging SLC35A2-2xFLAG plasmid into a Retrovirus system
6. Add the mixture dropwise to the cells from step 2 using a P1000 sterile pipette.
SECTION: Packaging SLC35A2-2xFLAG plasmid into a Retrovirus system
7. Incubate cells at 37 °C for 1440 min.
SECTION: Packaging SLC35A2-2xFLAG plasmid into a Retrovirus system
8. Replace media with 10 mL of fresh Growth Media and incubate cells for a further 1440 min at 37 °C.
SECTION: Packaging SLC35A2-2xFLAG plasmid into a Retrovirus system
9. Collect the culture media from step 8 (that now contains the retroviruses) and pass through a   syringe filter.
 


SECTION: Retrovirus infection (Transduction) and Selection of cells stably expressing SLC35A2-2xFLAG
10. Mix 5 mL of retrovirus infection media from step 9 with 5 mL of fresh Growth Media in a sterile 15 ml Eppendorf tube.
SECTION: Retrovirus infection (Transduction) and Selection of cells stably expressing SLC35A2-2xFLAG
11. Add Polybrene (10 mg/mL stock dissolved in MilliQ water, sterile filtered) to a final concentration of 10 µg/ml.
SECTION: Retrovirus infection (Transduction) and Selection of cells stably expressing SLC35A2-2xFLAG
12. Gently add to a   plate of HEK293 cells (or any cell line of interest) at ~60% confluency.
SECTION: Retrovirus infection (Transduction) and Selection of cells stably expressing SLC35A2-2xFLAG
13. Incubate at 37 °C for 1440 min.
SECTION: Retrovirus infection (Transduction) and Selection of cells stably expressing SLC35A2-2xFLAG
14. Change media to Growth Media and incubate for another 1440 min at 37 °C.
SECTION: Retrovirus infection (Transduction) and Selection of cells stably expressing SLC35A2-2xFLAG
15. To select cells stably expressing SLC35A2-2xFLAG, replace media with 10 mL of freshly prepared Selection Media.
 

SECTION: Retrovirus infection (Transduction) and Selection of cells stably expressing SLC35A2-2xFLAG
16. Change Selection Media every 24 hr for 4320 min - 7200 min to remove dead cells. After 5 days, cells stably expressing SLC35A2-2xFLAG should have reached 100% confluency.
SECTION: Retrovirus infection (Transduction) and Selection of cells stably expressing SLC35A2-2xFLAG
17. Cells can now be passaged and plated for experiments, or frozen down for long term storage in liquid nitrogen (Freezing media: growth media added with 10% v/v DMSO).