I'm glad you seem to be getting better results now! I used the Trex system to create four stable cell lines, and I learned a few things along the way.

1) Keep your tetracycline in the dark, and keep the lights as low as possible when you're working with Tet-containing media (i.e. keep the light off in the culture hood) - it's light sensitive.

2) Look at the manufacturer's instructions again - I used the Flip-in T-REx system (invitrogen Cat # K6500-01) and I had to use hygromycin and blasticidin together to select for positive clones. Blasticidin selects for the expression of the Tet Repressor gene, Hygromycin selects for insertion of your gene of interest, and Zeocin selects for an intact (not used yet) FRT site. Once your gene is inserted into the FRT site, the clone should be zeocin SENSITIVE, and Blasticidin and Hygromycin RESISTANT. To do the selection, I maintained the cultures in blasticidin and slowly increased the concentration of Hygromycin from 1uL Hygromycin per mL media to 3 uL Hygromycin per mL media over a period of 3 weeks (1uL/mL for one week, 2uL/mL for one week, and finally 3uL/mL for one week - by this time the control plate was all dead). If you increase Hygro too rapidly, even positive cells will die. I also had a untransfected control plate to be sure that the selection worked.

Good luck!!!

Tet-inducible systems can be very tricky!!! As David suggested, Tet-free media is very important. Also, how many colonies have you screened? Sometimes you have only one clone in 50 that has the right place of incorporation, resulting in a good system.

Hi Virginia,

I have several questions...

1. What is your current transfection efficiency (roughly)?

2. Are you using Optimem or PEI?

3. How big is your cargo?

4. What is your concentration of Tetracycline/Doxycycline that you are using for induction?

5. Have you characterized the repressed vs. induced cells via flow cytometry? If so, what is the fold increase in protein expression for induced vs. uninduced?

The problem that you have is most likely related to 1 (or more) of the five questions but I would like a little more information to be able to help you. I have quite a bit of experience in the generation of stable cell lines.

Dear Virginia,

I have not used the invitrogen system but I have generated tetracyclin inducible cell line. In my hand if you want to get a nice expression you have to combine Tetracyclin 2 ug/ml and Na butyrate (5mM) .. se my attached article

Article High-Level expression and purification of the human bradykin...

Virginia,

I've done Tet-inducible clones in the past.

- First, it's very important to select clones from your first transfections (using pcDNA6/TR) since this vector could get integrated in an transcriptionally active or inactive site. An easy way is:

- First select several clones, then transfect each clone with pcDNA4/TO/eGFP.

- If you see GFP without Tet, then the clone is bad.

- Select clones that have no GFP signal, then add Tet and follow (time course) the expression of GFP, select the clones that gave the signal level you are interested in (low or delayed expression might be good for toxic proteins).

- Then go back to the best clones and transfect them with your gene of interest.

David

Dear all,

Thank you very much for all your suggestions...I'll give you more info...and...I think I got encouraging results this week.

- I am using RT-PCR to test for protein expression because there is not an antibody commercially available for this protein. David, as a positive control for my reaction, I am using a stable clone that doesn't produce the Tet repressor, so you are right, I have really HIGH protein expression and I don't think the protein is toxic to the cells.

- I am putting a Sc. Cerevisiae gene in HaCaT cells so I don't think I need to worry about endogenous vs. recombinant protein expression.

- I already tried a transient transfection using both plasmids at the same time and as expected I saw some inducibility and considerable background of my protein, so I believe both plasmids are working.

- Joshua, the concentration of Tetracycline that I am using to induce protein expression is 10ug/ml and I wait 24h before harvesting. I am using Optimem and the transfection efficiency is roughly 20% using LTX.

- The GFP idea sounds good David, a lot of work but good. I do agree with you that I should pick clones after transfecting the TR plasmid but my PI told me not to because I would have to screen clones after transfecting the TO at the end anyway.

- Thanks for sharing your article Jacky, I will read it

- In the attached image you can see that clones 39 and 51 look promising (Yes...so far I have 72 clones total...How many do you usually screen Chrisna?) C1P15 to the far right is the positive control and HacatP12 to the far left is the negative control.

I would like to know your comments on the results, Do you think the difference between Tet- and Tet+ for clone 51 is good? average? bad? Should I go ahead and do experiments with these and check if I see a biological effect?

Hi, Virginia, Are you sure you select pCDNA6/TR positive clones with Zeocin and select pCDNA4/TO positive clones withblsticidin?

Dear Chufang, No I wrote it wrong, is the other way around, blasticidin for TR and zeocin for TO...thanks!

I'm glad you seem to be getting better results now! I used the Trex system to create four stable cell lines, and I learned a few things along the way.

1) Keep your tetracycline in the dark, and keep the lights as low as possible when you're working with Tet-containing media (i.e. keep the light off in the culture hood) - it's light sensitive.

2) Look at the manufacturer's instructions again - I used the Flip-in T-REx system (invitrogen Cat # K6500-01) and I had to use hygromycin and blasticidin together to select for positive clones. Blasticidin selects for the expression of the Tet Repressor gene, Hygromycin selects for insertion of your gene of interest, and Zeocin selects for an intact (not used yet) FRT site. Once your gene is inserted into the FRT site, the clone should be zeocin SENSITIVE, and Blasticidin and Hygromycin RESISTANT. To do the selection, I maintained the cultures in blasticidin and slowly increased the concentration of Hygromycin from 1uL Hygromycin per mL media to 3 uL Hygromycin per mL media over a period of 3 weeks (1uL/mL for one week, 2uL/mL for one week, and finally 3uL/mL for one week - by this time the control plate was all dead). If you increase Hygro too rapidly, even positive cells will die. I also had a untransfected control plate to be sure that the selection worked.

Good luck!!!

Hi Virginia, 72 clones are a good number...how many one should screen is a difficult question...it depends on time, money, number of viable colonies, etc. It basically comes done to "more is better"...

Two comments, I agree with David that you should screen after the first transfection as well. You will get better results if you do the second transfection with the best clone from the first transfection. Remember that you screen for different things after each transfection...

Second, have you tried inducing for a longer time than 24h? like 48 or 72 h? Also, did you perform a concentration series with the Tet to determine 10ug/ml as the optimal concentration?

I always say, having trouble in the lab is the best way to learn. Just one note on terminology use: RT-PCR is not used to test for protein expression, it is used to test for gene expression, you measure the gene's products, which are mRNAs, not proteins. Some people can argue that the presence of mRNA does not necessary mean protein production, and that can be true to a certain level. You can try adding a tag like Flag, His6, Myc, etc.

If you cannot detect your protein by western blot and if you face questions like: if you cannot detect the protein, how do you know the mRNA per se is what causes the effects? , be ready to (a) transfect cells with control sequence (the same mRNA product without start codon or kozak sequence, or (b) reverse sequence mRNA (I personally prefer the first choices).

TRex system is naturally leaky. High amount of TetR protein is critically required for suppression of the TO promoter before induction. So, I suggest you to select the best TR+ clone and transfect it for the second time with pCDNA6/TR. You may need more higher concentration of Dox to induce high level of GOI expression in the double-TR-transfected cells.

good luck with your experiments..

Transfecting twice the same cell line does not helps. The use of antibiotic selection is to kill the cells that did not receive the vector, but then the cells are keep in the antibiotic to kill the cells that over time loose the insert; If a cell has more than one insert, over time will lost them gradually, and the antibiotic allow us to keep the cells that maintain at list one active insert.

Thank you David

I think you are right, repeated transfections may not be helpful in long term, though it can increase TR copies..

But I think the level of TR expression is still the most important factor. Invitrogen has improved TRex system by using Flp-In Cell lines to avoid positional inactivation of TR and GOI.

If the TR+cells which can tolerate higher concentrations of blasticidin are selected, they may contain TR insertions in active chromatin positions. So possibly express more TR too!

Any way... I'm not sure!

It is supposed to be hygromycin for the TO plasmid,  blasticidin for the TR plasmid,  and Zeocin only prior to the TO/pcDNA5/gene of interest/FRT plasmid.  Zeocin should not be used after the final transfection unless you want to kill the cells for verification as a control.