Hi,I have been setting a over-expressed Jurkat cell lines for months but I cannot get a stable cell line. First ,I transfected 4 plasmids into 293T cells and collected the supernatant after 48h.Then I use this virus to infect my Jurkat cells for 24h and then change medium for next 1day. Then I use blasticidin 10ug/ml for selection. After 1-2 weeks' selection. I do WB to detect the expression for my protein.
Until now, everything is alright.
But only 1WEEK post selection, I hardly detect the difference between these selected jurkat and naive jurkat.
Does anyone have such experience in this experiment? Could you please give me some advice about lentivirus infection? THX.
Dear Liu Ying,
You can pick some single clones with higher expression level of you target proteins after blasticidin selection. Though lentiviruses can mediate long-time and stable expression of target gene, it is very common that some genes are silenced or lost after lentivirus infection. Single clone from single cell with obvious over-expression of target gene is indeed stable and a good solution to your problem.
Genemedi got a rich experience in lentivirus production and stable cell line construction, you could find more information on https://www.genemedi.net/i/lentivirus-packaging
Hope this may help you.
Hello, through what you found I can tell that you can't detect a big difference between your selected jurkat and naive ones because of two possible mistakes: the first one is you have to quantify your virus in tour supernatant to be sure that the transfection was properly done and your virus is produced properly. The second one is the dose of the blasticidin that you are using for selection, I guess you can optimize it. Good luck
If you see stable (surviving) Jurkat cells after 1-2 weeks then at least you know that (1) you have virus and (2) it is integrating into the Jurkat cells. This is assuming that you have properly selected the 10 ug/mL dose of blasticidin that you use for selection (i.e., at least that wild-type Jurkat cells do not survive this selection. There are also other factors to consider when selecting the dose.).
A few questions to consider:
Blasticidin is a very potent selection agent. You should see virtually no Jurkats surviving after 48 hours if you do a lymphocyte separation. Hope that helps....
Hi Liu Ying,
First, better check your expression vector by simple transfection in HEK 293.
1. Did you check the viral functional titer? I mean did you infect your viral particles to HEK293 cell line with 10 fold serial dilution and check the target protein expression levels?
- With this you will get to know whether your viral particles have packaged your expression vector or not.
2. 8 microgram/ml concentration of Polybrene works well. (worked for me with Jurkat-T cells, lentiviral method)
3. Spinoculation or Incubation methods with different MOI will definitely work.
4. The best thing what you can do is-
Incubate 0.2-0.5 million cells per 100-200 microliter respective growth media containing polybrene (8 microG/ml) and target viral particles in a falcon or eppendrof. Incubate this mixture(cells+growth media+Polybrene+Virus) overnight in CO2 incubator.
Next day wash the mixture and top up fresh media containing polybrene until 24 hrs. later wash the cells and go for selection and some cells expand and check the expression.
Hope this would surely help...
Try to transduct Jurkat with GFP to optimize the production of stable line. It will make the whole thing much easier. My experience - I used spinoculation. GFP was under PGK promoter. GFP appeared only about a week after trancduction, but then the cells exhibited green light over a month (until I killed the culture). Maybe your problem is too little transduction efficiency - there is still too much naive cells in your culture that dilute infected cells with every passage. Try to use higher titer or not to remove virus containing medium until the next passage.
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