The way we usually do it is through retroviral gene transfer which is done using plasmid that contains IRES-GFP or IRES-YFP, gag, pol and env for retroviral transfection into 293T cells line (using calcium phosphate transfection protocol), then taking the sups from these cells and introducing the sups onto the cells of interest followed by spin infection of those cells (1,5h at the max speed, spin infection can be done in 6 well plate or 10cm plate). If you need more info let me know

I would like to have a little more detail about the later part, where u have a clone with hygro.

but I can say that the 200microgm/ml of G418 is low concentration to select the stable clones, with this concentration there is a chance to have false colonies. since you mentioned, u have done a Kill curve, so that would be helpful, if you can share some result of that.

I would transfect the cells with TetR or Tet ON plasmids, in a P100, grow for 48 hrs, then add G418 in 1/2/3 microgm /ml. you will see the effect in 2-4 days, drop the G418 accordingly. You mentioned you had a mock cells too, and they all died (that will not ensure the other plate are not false positive, because the time 2 weeks is quite good enough to develop the resistance).

Also with the plasmid transfection, it gets randon insertions and DNA gets fragmented too, so u might have G418 resistance but not TetR element in there.

I dont have any specific answer to your question since I have not used MCF10A specifically.

We have done this with mammary epithelial cell lines (16A5 and 76N-TERT) - using retroviral infection in two steps. Check our publications in JBC (Duan L et al 2010; Nag A et al. 2007). Once you have read through, you can email me directly for additional info and I can connect you to people that actually did the experiments. Best, H

Hi Chris,

I have been working with a similar system and had similar troubles (have used clontech and the invitrogen gateway (pDEST-DOX)). We have been using esophageal adeno lines (also epithelial). You seem to have the right approach, much as we thought we did, but here are some things we did that made a difference. Obviously there will be cell type specific affects that might be hard to get around. We have found liposomal transfection better than electroporation, particularly good results with Lipofectamine 2K (we tried about 8 different lipo-based reagents, including Mirus, gene juice, original lipofectamine, metafectine, and all gave poor results except for L2K). We have found that reverse transfection is best. We usually limit to transfection with 5 ug DNA and replate on 100 cm2 dishes. After liposomal transfection we only leave our cells 24 h before beginning selection with G418. We have found that some of our cell lines are highly resistant to multiple transfections. The colonies you are picking after selection may be quite resistant to multiple transfections? We find no difference in transfection efficiency or colony number whether a plasmid was linearised or not. But, we have also found that the cells don't like to take up too much plasmid DNA, we find toxicity based on the amount of DNA transfected, particularly on multiple transfections, even with scrambled or vector controls. We have also found, but haven't really been able to explain it, that introduction of a second plasmid (or more plasmis) blunts expression of another. We've also found that when selecting with multiple antibiotics, such as G418 and hygromycin, their sensitivity profiles differ when selecting double positive clones compared with single positive clones...unfortunately you may need to re-titrate your G418 kill curves in the TRE stable expression line.

Not sure if any of this helps you, but it all sounded very familiar.

Stephen

PS, I lost a lot of hair during those experiments, but maybe that was just a coincidence!!!

Hi guys - thanks for your comment and suggestions.....

@Dzana/Hamid - So yes, lentiviral constructs seem to be what most people have used in the literature, however my boss is very anti-lentivirus at the moment (health and safety issues/paperwork etc) so that is on the back-burner at the moment!

@Stephen - Thanks for your suggestions. Yes - I think I tried lipofectamine 2K (aswell as LTX) and the cells didnt seem to like it (although I was doing O/N transfections rather than 6 hours). From my studies we found Mirus TransIT LT to be the best for transfecting cells (I think it is actually just Fugene6 under a different name!). However I didn't try a reverse transfection - I will give it a go!

I actually managed to get the Tet-responsive stable cell line by transfecting with the Mirus reagent, and can transiently transfect the Tet-ON plasmid using the same reagent into this line and get lots of transfected/expressing cells. Suggesting that multiple transfections are not an issue with these cells (Although they are quite hard to transfect in the first place - hence trying electroporation).

I did think that selecting with multiple antibiotics may alter the sensitivity of the cells, but I managed to get GFPC1 stably transfected cells in the Hygro resistant cell line, when selecting with the same concentration of G418 + HygroB as I am for the TetON transfected cells.

People have told me (although I havent managed to find anything in the literature backing it up) that TetR expression is downregulated when stably expressed - could it be that the levels of expression are too low to detect? Then again, people have also said that only 1-2 copies of TetR are needed to be expressed in order to drive tet-responsive elements and overexpression of the GOI. My boss has suggested using Ca phosphate transfection in order to get multiple copies of the driver into the nucleus - although the murky world of Ca phosphate transfection doesnt fill me with much excitement......

Stephen - did you devise your own reverse transfection protocol with L2K? Can't seem to find anything about it in invitrogen's manuals!

Hi Chris,

With the L2K we only transfect for 6 hours, the O/N tends to cause a lot of toxicity. We have been doing a lot of clonogenic assays following overexpression and radiation treatment, so we grow the cells for about 10-12 days post transfection (even after a transient). We've found that liposomal reagents on their own cause long term toxicity in cells, i.e. the cells look fine for the first few days but they die off after about 5-7 days (not sure of the exact form of cell death, but their colony forming potential is quite poor as a result). We've gotten around this by plating at higher numbers post transfection. I think on subsequent transfections (perhaps even with electroporation, not just liposomes) you actually get more toxicity than in first transfections.

I still wouldn't dismiss the possibility that the G418/Hygro double treatment isn't a problem! We've found that different genes in the same plasmid backbone can still alter sensitivity.

If it were me I'd stay away from the Ca phosphate transfections a little longer. Try the reverse transfection first. It's essentially similar to the electroporation method, but uses liposomes (it also cuts 24 h off the set-up time). With the reverse transfection the cells are liposomally-transfected in suspension, as opposed to transfecting cells in a dish at 80%+ confluence. Transfecting epithelial cells in suspension with liposomes dramatically increases their surface area, and we find much better transfection efficiency. The procedure is below. You'll have to modify this accordingly for your own experimental setup

Procedure:

1. Harvest cells in exponential phase by trypsinisation.

2. Resuspend cells at 1x10e5 cells/mL in antibiotic-free Opti-MEM I medium.

3. Dilute transfection reagent (5-9 uL) in 300 uL ab-free, serum-free Opti-MEM per well/dish to be transfected. Incubate 10 min at RT.

4. Dilute DNA (5 ug) in 300 uL ab-free, serum-free Opti-MEM medium. Combine this solution with the former (600 uL total). Mix gently and incubate for 15 min at RT.

5. For a 6-well plate, dispense the 600 uL DNA/liposome complex into a well. A volume of 2.4 mL cell suspension is added to the well (final density 2.4x10e5 cells/well). Gently agitate for 30 s to mix.

6. Incubate at 37C, 5% CO2, humidified.

7. Begin selection 24 h post tranfection.

Thanks Stephen - Ill give it a go!

So you did you selection for stable cells in 6 well plates? How many colonies did you get per well? I was thinking of doing it in 10cm dishes - would I just scale up (i.e. approx 4 times as much of everything?)

No problem.

In a 6-well plate we usually get around 15-20 colonies per well. I usually tend to do my selections on 100 cm dishes, where I'll get 100+ colonies (usually get about 1 in 15 to 1 in 20 escapees). You don't necessarily have to scale everything up in parallel, sometimes the selection and clone numbers work better using less reagent and more cells.

and what kind of percentage of the colonies express? Do you pick and screen them all?

We get quite high expression rates (>90%)...we find very few that escape the selection, and those that do usually form slightly smaller colonies.

Unfortunately I can not bring anything new. I buy MCF10A at the ATCC and used for toxicological studies. Proceed in accordance with the protocol recommended by the ATCC

Unfortunately I can not bring anything new. I buy MCF10A at the ATCC and used for toxicological studies. Proceed in accordance with the protocol recommended by the ATCC

Sorry Stephen - more Q's:

- So if im scaling up to 10cm dishes, then the 600ul TF mix is not going to cover the plate is it? Is that an issue? Or when the cell suspensions are added does it all just mix together?

- Based on my calculations, to scale up number of cells for 10cm dish it would be somewhere around the 1-1.5x10e6 cells mark (based on surface area)

- I guess 6 hours is long enough for the cells to attach to the plate before swapping out the TF mix media - do you incubate overnight in normal culture media (i.e. plus serum and supplements - I dont use pen/strep in my media) and then the next day change for normal media +G418??

- Your stable cells are not constitutively expressing are they? They are under Tet/dox control yeah? When you screen for expression of TetR in the driver line (Which is what im attempting to make) do you transiently transfect in a TRE-reporter plasmid and induce with dox? Our reporter plasmids are biscistronic with a IRES site between our GOI and GFP. Speaking to clontech they suggest pre incubating the clones with dox before TF to ensure that when the TRE-element is inside the cell, the dox can bind straight away and increase levels of expression (since they say that the GFP after the IRES signal will not be expressed aswell as the gene before it). Have you done this?

It's not important for the 600 ul of transfection reagent to cover the base of the well/dish. When the cell suspension is added it all just mixes together, which is fine.

Yes, those scale up numbers are fine for that size dish.

Usually epithelial cells will have adhered within 4 hours of seeding, so removing the medium after 6 hours is fine. I incubate overnight in complete medium, also without P/S, and after the 24 h period switch to complete medium plus G418.

My cells are not constitutively expressing. They are dox induced. Yes, the TRE is transiently expressed and Dox induced. Pre-tx with the dox is a good idea. We've not had to do this as far as I'm aware. Although, this might potentially cause toxicity issues. With regard to their explanation of why the GFP might not be expressed as well as the GOI is not clear. I know there can be issues using bicistronic reporter constructs, but if it's commercially available it should work fine!

I hope this is helping and not just making things worse for you!!!

Nope - all suggestions are greatly appreciated. Im going to give it a go next week and let you know how it goes!

So, did you ever get the stables?  I am currently attempting stable integration with the Tet-On system in MCF10As and am exasperated.  I have tried all the tricks, and am going to switch to transduction (not preferred for this project).  Please advise.

Thanks!

can some one help me in finding SNPs in MCF10A cell line..