TITLE: Organelle Tag Introduction in Mouse Embryonic Fibroblasts (MEFs)
AUTHORS: Matthew Taylor, Francesca Tonelli, Dario R Alessi

[DESCRIPTION]
We describe here a method to generate MEFs stably expressing a lysosome localized TMEM192-3×HA fusion protein (“LysoTag”), or a Golgi localized TMEM115-3×HA fusion protein (“GolgiTag”). The fusion proteins mark lysosomes or the Golgi apparatus, respectively, with a triple HA epitope, enabling the rapid isolation of intact tagged organelles from cells using an anti-HA antibody. The purified organelles can then be analysed with a variety of techniques including immunoblotting analysis and proteomic, lipidomic, or metabolomic analysis.This protocol can be adapted to introduce organelle tags in commonly cultured cells, such as HEK293 and A549 cells. For MEFs, we recommend performing a Simian Virus 40 (SV40) immortalisation of primary cells prior to the introduction of the organelle tag, which is also described here.

[STEPS]
SECTION: 1.1 Packaging the SV40 large T antigen plasmid into a Retrovirus system
1. 1.1.1) Grow HEK293FT cells to 60% confluency in Growth media in a 10 cm Petri Dish.
SECTION: 1.1 Packaging the SV40 large T antigen plasmid into a Retrovirus system
2. 1.1.2) Prepare a transfection mix in a sterile 1.5ml Eppendorf tube, containing:
3.8 µg pCMV Gag/Pol plasmid
2.2 µg pCMV VSV-G plasmid
6 µg pBabeD SV40 Large T Antigenplasmid
36 µl 1mg/ml PEI Max 40K
500 µl OptiMem
4. 1.1.4) Add the mixture dropwise to the cells from step 1.1.1 using a P1000 sterile pipette.
SECTION: 1.1 Packaging the SV40 large T antigen plasmid into a Retrovirus system
3. 1.1.3) Mix by vortexing and incubate at room temperature for 20 mins.
5. 1.1.5) Incubate cells at 37°C for 24 h.
6. 1.1.6) Replace media with 10 mL of fresh Growth Media and incubate cells for a further 24 h at 37°C.
7. 1.1.7) Collect the culture media from step 1.1.6 (that now contains the retroviruses) and pass through a 0.45 µm syringe filter. 
Note: The retrovirus infection media from step 1.1.7 can be used immediately (Step 1.3.1) or stored at -80°C for subsequent use.
SECTION: 1.2 Packaging the Organelle Tag (LysoTag or GolgiTag) plasmid into a Lentivirus system
8. 1.2.1) Grow HEK293FT cells to 60% confluency in Growth media in a 10cm Petri Dish.
SECTION: 1.2 Packaging the Organelle Tag (LysoTag or GolgiTag) plasmid into a Lentivirus system
9. 1.2.2) Prepare a transfection mix in a sterile 1.5ml Eppendorf tube, containing:
3.8 µg pCMV Gag/Pol plasmid
2.2 µg pCMV VSV-G plasmid
6 µg pLJC5 TMEM192-3xHA (LysoTag), or pLJC5 TMEM115-3xHA (GolgiTag), or pLJC5 HA-Empty (control)
36 µl 1 mg/ml PEI Max 40K
500 µl OptiMem
SECTION: 1.2 Packaging the Organelle Tag (LysoTag or GolgiTag) plasmid into a Lentivirus system
10. 1.2.3) Mix by vortexing and incubate at room temperature for 20 mins.
SECTION: 1.2 Packaging the Organelle Tag (LysoTag or GolgiTag) plasmid into a Lentivirus system
11. 1.2.4) Add the mixture dropwise to the cells from step 1.2.1 using a P1000 sterile pipette.
SECTION: 1.2 Packaging the Organelle Tag (LysoTag or GolgiTag) plasmid into a Lentivirus system
12. 1.2.5) Incubate cells at 37°C for 24 h.
SECTION: 1.2 Packaging the Organelle Tag (LysoTag or GolgiTag) plasmid into a Lentivirus system
13. 1.2.6) Replace media with 10 mL of fresh Growth Media and incubate cells for a further 24 h at 37°C.
SECTION: 1.2 Packaging the Organelle Tag (LysoTag or GolgiTag) plasmid into a Lentivirus system
14. 1.2.7) Collect the culture media from step 1.2.6 (that now contains the lentiviruses) and pass through a 0.45µm syringe filter. 
Note: The retrovirus infection media from step 1.2.7 can be used immediately (Step 1.4.2) or stored at -80°C for subsequent use.
SECTION: 1.3 Viral transduction and selection of MEFs stably expressing SV40
15. 1.3.1) Mix 5 ml of retrovirus infection media from step 1.1.7 with 5 ml of fresh Growth Media in a sterile 15 ml conical centrifuge tube.
SECTION: 1.3 Viral transduction and selection of MEFs stably expressing SV40
16. 1.3.2) Add Polybrene to a final concentration of 10 µg/ml and mix by vortexing.
SECTION: 1.3 Viral transduction and selection of MEFs stably expressing SV40
17. 1.3.3) Add dropwise to a 10 cm plate of primary MEFs at ~70% confluency. 
SECTION: 1.3 Viral transduction and selection of MEFs stably expressing SV40
18. 1.3.4) Incubate at 37°C for 24 h. 
SECTION: 1.3 Viral transduction and selection of MEFs stably expressing SV40
19. 1.3.5) Change media to Growth Media and incubate for another 24 h at 37°C.
SECTION: 1.3 Viral transduction and selection of MEFs stably expressing SV40
20. 1.3.6) To select cells stably expressing SV40, replace media with 10 ml of freshly prepared Selection Media containing 200 μg/ml Hygromycin.
Note: 
MEFs that have not been infected should be included as a control for the efficiency of the selection agent.
MEFs that have not been successfully transduced should start dying 24 h after the addition of selection media.
SECTION: 1.3 Viral transduction and selection of MEFs stably expressing SV40
21. 1.3.7) Change Selection Media every 24 h for 3-5 days to remove dead cells. After 5 days, cells stably expressing SV40 should have reached 100% confluency. 
Cells can now be passaged and plated for further experiments (Step 1.4.1), or frozen down for long term storage in liquid nitrogen (Freezing media: growth media added with 10% v/v DMSO).
SECTION: 1.4 Viral transduction and selection of MEFs stably expressing Organelle Tag
22. 1.4.1) Plate SV40 immortalised MEF cells from Step 1.3.7 in 10 cm dishes to give a 70% confluency the following day.
SECTION: 1.4 Viral transduction and selection of MEFs stably expressing Organelle Tag
23. 1.4.2) Mix 5 ml of lentivirus infection media from step 1.2.7 with 5 ml of fresh Growth Media in a sterile 15 ml conical centrifuge tube.
SECTION: 1.4 Viral transduction and selection of MEFs stably expressing Organelle Tag
24. 1.4.3) Add Polybrene to a final concentration of 10 µg/ml and mix by vortexing.
SECTION: 1.4 Viral transduction and selection of MEFs stably expressing Organelle Tag
25. 1.4.4) Add dropwise to cells from Step 1.4.1. 
SECTION: 1.4 Viral transduction and selection of MEFs stably expressing Organelle Tag
26. 1.4.5) Incubate at 37°C for 24 h. 
SECTION: 1.4 Viral transduction and selection of MEFs stably expressing Organelle Tag
27. 1.4.6) Change media to Growth Media and incubate for another 24 h at 37°C.
SECTION: 1.4 Viral transduction and selection of MEFs stably expressing Organelle Tag
28. 1.4.7) To select cells stably expressing the organelle tag, replace media with 10 ml of freshly prepared Selection Media containing 200 μg/ml Hygromycin and 2 μg/ml Puromycin.
Note: 
MEFs that have not been infected should be included as a control for the efficiency of the selection agent.
MEFs that have not been successfully transduced should start dying 24 h after the addition of selection media.
SECTION: 1.4 Viral transduction and selection of MEFs stably expressing Organelle Tag
29. 1.4.8) Change Selection Media every 24 h for 3-5 days to remove dead cells. After 5 days, cells stably expressing the organelle tag should have reached 100% confluency. 
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).

Note: We recommend confirming the expression of organelle tags by detection of HA-Tag by performing quantitative immunoblotting analysis as described in dx.doi.org/10.17504/protocols.io.bsgrnbv6.