Hi,

In order to make stable HEK293 cells, you need to transfect your HEK293 cells using the available transfection agent (we generally use Xtreme Gene or Lipofectamine 2000) as per the manufacturers protocol.

To do so, you can follow the following steps:

Note: For each well plate format, the amount of DNA to be added to the transfection reagent will be mentioned in the manufacturers datasheet. 

Hope this helps.

Amir

pcDNA 3.1 has Neomycin as a reporter cassete. If you want to create an stable cell line from HEK-293, first check that these cells are not resistant to Neo performing a killing curve assay. If this works then transfect the cells with the pcDNA 3.1 and then add Neo to the grow media. 

When you do this, you will have multiple colonies in your plate/wells, pick those colonies and expand them in a growth media containing Neo and I guess that you would have something else within the vector pcDNA, so analyze these clones for your gene of interest.

Hi,

In order to make stable HEK293 cells, you need to transfect your HEK293 cells using the available transfection agent (we generally use Xtreme Gene or Lipofectamine 2000) as per the manufacturers protocol.

To do so, you can follow the following steps:

Note: For each well plate format, the amount of DNA to be added to the transfection reagent will be mentioned in the manufacturers datasheet. 

Hope this helps.

Amir

Hello,

In addition to the above mentioned points, do note that there are two types of pcDNA3.1 vectors available:

pcDNA3.1(+) and pcDNA3.1(-)

Based on the antibiotic resistance available in your vector backbone, determine a kill curve by using an increasing concentration of available antibiotic.

The concentration at which you observe absolute cell death in 7-10 days is the concentration you are required to use for selection of population containing your gene of interest.

Also note, HEK293 is a hard to transfect cell line as stated by a several transfection reagent booklets, it would be advisable to use transfection reagents like Lipofectamine 3000 (instead of 2000/1000), Xtreme Gene, or Superfect.

Good luck!

Hi,

I would suggest to have a gfp tag for your protein, transfect , select with appropriate antibiotics and go for FACS sorting. If you don't have antibiotic resistance in your plasmid, co-transfect (1:10) with puromycin vector.  I used Xtremegene 9 (3:1)  as it is less toxic.  Transfect in serum free condition and add serum alone after 5 hours of transfection, without removing the transfection complex, until they are ready for splitting. Even after FACS sorting, the expression is not very stable for some cell lines and so, I have moved on to viral transduction.

Usually the HEK293T cells are the ones resistant to G418. Nonetheless the «plain» HEK293 cells are still hard to work with. I tried using them and they need drug concentrations of ~900-1000μg/ml. If you still haven't got the construct cloned in the pcDNA3.1 there is a version of the vector containing Zeocin resistance. Used that one in establishing stable lines and worked just well. In any case you need to start with a kill curve for your drug!! I usually choose the time point of 5 days to determine the concentration to use. 

After transfecting the cells I usually culture them in media containing the drug for 5 days. The remaining cells are tripsinized and transferred to 10cm dish in a very low concentration (1000 cells per plate) and continue culturing them in the presence of the drug. A few days later you will see colonies forming in the plate, transfer them to 96 well plate and continue as mentioned above. 

Have worked with both the cell line and the vector to obtain stable lines, if you need anything else contact me and will gladly help you. Good luck!! 

P.s. These cells are very easily transfected so you won't need any commercial transfection reagent. I use 2xBES at pH 7,0 and works just fine for me (~70% transfection efficiency) 

Transfection of a plasmid into a cell leads almost to a transient expression of your gene of interest! Stable integration of the plasmid DNA is poor event and can change a lot things in your cells. All other scientist here are right that you can try to select cells that have stably integrated the plasmid and therefore for your gene of interest and respective markers (antibiotic resitant markers or fluorescent proteins), but this needs time for checking different clones for gene expression and for growing the clones. During this procedures e.g. extented cell expansion as well as under long term antibiotic selection, cells will change in general enormously. Furthermore, you have to add all the time the antibiotics! Otherwise the cells will either reduce the expression of the gene of interest or disintegrate the respective DNA over time! When cells are established, you have to compare HEK cells with the empty plasmid and cell with the plasmid including gene of interest! Viral transduction models are more integrative and the better choice as method for stable integration and constitutive expression of your gene of interest. Good luck!

I totally agree with Peter, but this is something you won't be able to do with pcDNA3.1. you need to obtain a good lentiviral expression vector and clone your gene of interest there. If you are considering to do something like that, how about using an inducable system (tet-on) 

I agree with Peter that stable integration of plasmid DNA can affect a lot of things in a cell. Initially, I used to do colony picking and found to have different properties between colonies, despite similiar expression level. It could be a clonal effect, since the population is from a single colony, mono clonal. So, instead of colony picking, we started doing FACS sorting to have polyclonal population and to avoid this clonal effect. With FACS , it is possible to sort high or medium expressing cells and also to resort, if they start to loose some expression. If there is atleast one sticky end ligation site, it should'nt be difficult to move it form pcDNA 3.1 to viral vector. Viral transduction is efficient and has the advantage to choose a range of host cell lines for stable transfection.

MY lab has been using HEK cells to generate viral media for transduction into other cell lines. we are able to achieve  at least 95% transfection efficiency. what, we do is to grow HEK cells in complete media (media with serum and antibiotics) in  10 cm plates for overnight to achieve 80% confluency. 2 hours before transfection we remove the media, wash once with PBS and then add media without antibiotics but containing the normal 10% serum. We use Polyethylenimine (PEI)( commercial availble in powdered form) which could be dissolved with distilled water and filtered. we use PEI:DNA in  a ration of 5:1. for 10cm plates, we use 10.5ug of DNA and 52.5 ug of PEI. As I said, we are able to achieve at least 95% transfection efficiency with this protocol. Moreover, Polyethylenimine (PEI), is very very cheap compared to other transfection reagent. With such a high transfection efficiency, you can proceed to do your clonal selections as suggested above. do not hesistate to contact me if u need more details on this protocol.wish u the best.  

HELLO

Dear all,

I am working to make a stable cell line of Rabbit kidney 13 cells (RK13) expressing one of the herpes virus proteins, I have determined the killing dose of Geneticine, and I am going to transfect my cloned pcDNA3.1(+) into cells soon.

My inquiry is about picking of the resistant cell colonies after transfection. If the plate contain some colonies, how can I collect each one separately. In case of trypsinization, I think the adherent cells will be dissociated and  mixed together and I will not be able to take separate colonies. Would you help me in understanding how to collect single colonies?.

Best regards

Hi Yassien, 

I really don't know if the cell line you are using is one of the tough ones for splitting. Anyways one of the best (maybe the best) ways to isolate clones is to use some plastic cylinders. These have small diameter and can be attached on the plastic dish using grease (Vaseline for example). Each cylinder contains one colony that can then be trypsinized and transferred separately to a well. I am sending you a link with the description and a picture to make it easier for you to understand. 

Good luck 

http://www.sciencelab.com/page/S/PVAR/23772/60-378470000

I would suggest you to use other vector that allows the integration of your gene of interest in the genome like pBABE,  pMSCV.....

best regards

When I did like him, using pcDNA3.1 flag1 +gene of interest and tranfected it into  HEK293, I check with Western blot of my cell lysate for nearly one month after transfection. However, I could not see the target band in WB (all control, empty vector and vector contain my target gene couldn't visible. The antibody is specific for my target protein as determined previously. Attached file is my SDS-PAGE gel and WB. My target protein is 15kDa., the position is MW, time point after transfection, 4 days later and 1 month. Please let me know the possibility. Thank you so much for your help!

I am not surprised to see no expression long time after transient transfection. Protein expression through transient transfection normally last less than one week. In HEK293 or 293T cells, you will observe the protein expression as short as 8 hours after transfection. I suggest you check your protein expression at 24h, 48 hours post transfection instead of 4d and 1m. If you target protein is not stable, you'd better add some protease inhibitor in the media for 2-4 hours before sample collection to enhance the protein expression. You also need to confirm your transfection is great using GFP plasmid. Normally, 293T cells can be transfected as high as >95% efficiency.

To get a stable cell line in 293 cells series, you'd better subclone you gene in another plasmid with selection marker instead of Neomycin or G418. If you heat to do that, there is one way to do the selection. Cotransfect you plasmid with another vector carrying hygromycin or puromycin resistance gene and select with hygromycin or puromycin. Then pick up single colony and check which clone express your target gene. Keep expanding that clone with hygro or puro media. this method only works for single colony selection and doesn't work as stable cell pool.

Please, take into consideration that a DNA vector, a transfection reagent, expression of an antibiotic resistance (trans)gene, expression of a reporter (trans)gene, and selection by acute/chronic antibiotic treatment may evoke cellular responses that affect the biochemical processes under investigation. In the following review, we demonstrate that an assumption that empty vector-transfected cells preserve the cytogenetic and phenotypic characteristics, and represent the adequate control in transfection experiments is not universally valid. We exemplify a number of studies, including on HEK293 cells, which reported obvious genomic, transcriptomic and phenotypic changes of tumor cells after transient/stable transfection of an empty vector. Finally, we conceptualize that the diverse experimental manipulations (e.g., transgene overexpression, gene knock out/down, chemical treatments, acute changes in culture conditions, etc.) may act as a system stress, promoting intensive genome-level alterations (chromosomal instability, CIN), epigenetic and phenotypic alterations, which are beyond the function of manipulated genes.

Please see the following manuscripts for details:

Stepanenko AA, Heng HH. Transient and stable vector transfection: Pitfalls, off-target effects, artifacts. Mutat Res. 2017 Jul;773:91-103. doi: 10.1016/j.mrrev.2017.05.002.

Stepanenko A, Andreieva S, Korets K, Mykytenko D, Huleyuk N, Vassetzky Y, Kavsan V. Step-wise and punctuated genome evolution drive phenotype changes of tumor cells. Mutat Res. 2015 Jan;771:56-69. doi: 10.1016/j.mrfmmm.2014.12.006.

Stable transfection of HEK293 is most improved by using HEK293-specific transfection reagents (e.g. https://altogen.com/product/hek-293-transfection-reagent-epithelial-kidney-cells/). Several rounds of selection can help you improve the homogeneity of your cell population, while transduction-based techniques can offer higher efficiency at the expense of cell viability and other extraneous effects. Plasmid size is a factor that can negatively affect efficiency, so using complex condensers can help you improve overall results.

Hi, Elamurugan Appavoo!

To make stable HEK293 cells, we always transfect HEK293 cells with the available transfection agent (we generally use Lipogene, comparable to lipofectamine 3000, but with fewer cytotoxic effect. You could find the protocol of this product attached.

Take a 6-well plate as an example, the detailed protocols are as follows.

1) Seed cells one day in advance to make sure the confluency can be 70%-80% before transfection.

2) Transfect the required amount of DNA using the LipoGene as the manufacturers protocol suggested.

3) Change the media 6 hours post-transfection to achieve higher transfection efficiency.

4) 48 hours post-transfection, select cells with antibiotic in your vector backbone. The overexpression efficiency can be determined with Q-PCR or Western blot.

5) Then you should perform you experiments as soon as possible, for this method is just for transient cell lines.

If you want to establish stable cell lines, virus mediated transfection is needed, Lentivirus is really an efficient way to generate stable cell lines. Integration into host cell genome, it mediates long-term and stable expression of exogenous genes with a simple system for vector manipulation and production. We got a rich experience in lentivirus production and titration, you could find more information in the protocol attached.

Hope this may help you.