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General

We would recommend adding a stop codon after your gene sequence, in front of the epitope tag so that the tag is not expressed. 

Translation initiation will occur at the first ATG encountered by the ribosome, although in the absence of a Kozak sequence, initiation will be relatively weak. Any insert downstream would express a fusion protein if it is in frame with this initial ATG, but levels of expressed protein are predicted to be low if there is a non-Kozak consensus sequence. If the vector contains a non-Kozak consensus ATG, we recommend that you clone your gene upstream of that ATG and include a Kozak sequence for optimal expression. 

No; neomycin is toxic to mammalian cells. We recommend using Geneticin® (a.k.a. G418 Sulfate), as it is a less toxic and very effective alternative for selection in mammalian cells. 

Here are possible causes and solutions:

  • Try the control expression that is included in the kit
  • Possible detection problem:
    • Detection of expressed protein may not be possible in a transient transfection, since the transfection efficiency may be too low for detection by methods that assess the entire transfected population. We recommend optimizing the transfection efficiency, doing stable selection, or using methods that permit examination of individual cells. You can also increase the level of expression by changing the promoter or cell type.
    • Expression within the cell may be too low for the chosen detection method. We recommend optimizing the detection protocol or finding more sensitive methods. If the protein is being detected by Coomassie/silver staining, we recommend doing a western blot for increased sensitivity. The presence of endogenous proteins in the lysate may obscure the protein of interest in a Coomassie/silver stain. If available, we recommend using a positive control for the western blot. 
  • Protein might be degraded or truncated: Check on a Northern.
  • Possible time-course issue: Since the expression of a protein over time will depend upon the nature of the protein, we always recommend doing a time course for expression. A pilot time-course assay will help to determine the optimal window for expression.
  • Possible cloning issues: Verify clones by restriction digestion and/or sequencing.

Here are possible causes and solutions:

  • Detection method may not be appropriate or sensitive enough:
    • We recommend optimizing the detection protocol or finding more sensitive methods. If the protein is being detected by Coomassie/silver staining, we recommend doing a western blot for increased sensitivity. The presence of endogenous proteins in the lysate may obscure the protein of interest in a Coomassie/silver stain. If available, we recommend using a positive control for the western blot. 
  • Insufficient number of clones screened: Screen at least 20 clones.
  • Inappropriate antibiotic concentration used for stable selection: Make sure the antibiotic kill curve was performed correctly. Since the potency of a given antibiotic depends upon cell type, serum, medium, and culture technique, the dose must be determined each time a stable selection is performed. Even the stable cell lines we offer may be more or less sensitive to the dose we recommend if the medium or serum is significantly different.
  • Expression of gene product (even low level) may not be compatible with growth of the cell line: Use an inducible expression system.
  • Negative clones may result from preferential linearization at a vector site critical for expression of the gene of interest: Linearize the vector at a site that is not critical for expression, such as within the bacterial resistance marker.

We recommend checking both the cellular lysate and the medium for presence of the protein of interest. While a given secretion signal sequence will work for secretion of most proteins, it is not guaranteed for all proteins. Hence, it is necessary to experimentally determine if the protein of interest is being secreted properly. 

For issues related to restriction enzyme cloning, please visit our Restriction Enzyme Cloning Support Center. For issues related to cloning into a TOPO® vector, please visit our PCR Cloning Support Center and for problems related to cloning into a Gateway® vector, please visit our Gateway Recombination and Seamless Cloning Support Center.

Inducible/Regulated Expression—T-REx™, Flp-In™ T-REx™, and GeneSwitch™ Systems

Almost all lots of FBS contain tetracycline, because FBS is generally obtained from cows that have been fed a diet containing tetracycline. If cells are cultured in medium containing FBS that is not reduced in tetracycline, there may be low basal expression of the gene of interest in the absence of added tetracycline. In such cases, we recommend purchasing tetracycline-reduced FBS from our Gibco® Cell Culture Division. To be qualified as tetracycline-reduced, these lots must contain below 19.7 ng/mL tetracycline (which happens to be the assay detection limit).

Note: The binding constant for Tet-repressor protein with tetracycline is 3 nM. Assuming that the medium contains 10% serum, a serum tetracycline concentration of 19.7 ng/mL is equivalent to 4 nM tetracycline. Thus, keep in mind that it is possible to get basal level expression even from tetracycline-reduced FBS.

Targeted Expression—Flp-In™ and Jump-In™ Systems

Before giving up, we would suggest that you try using the pFRT/lacZeo2 vector to generate your host cell line. This vector contains a truncated version of the SV40 promoter driving the lacZ-Zeocin fusion. Use of this vector facilitates the isolation of clones that have integrated the vector near enhancer elements in the genome, thus resulting in higher levels of expression of the gene of interest.

The Flp-In™ 3T3 cell line is derived from NIH3T3 cells, which are mouse fibroblast cells. The CMV promoter is known to get silenced over time in murine cell lines and hence we would recommend using a Flp-In™ expression vector with a non-CMV promoter in these cells, such as the pEF5/FRT/V5-D-TOPO® vector or the pEF5/FRT/V5-DEST vector.

We have observed in-house that in cells where the FRT site has integrated into a very transcriptionally active locus in the host cell genome (seen more commonly in Flp-In™ CHO and Flp-In™ 293 cells but can also happen in Flp-In™ 3T3 cells and any other Flp-In™ host cell line), there is some "read-through" transcription and translation of the lacZ-Zeocin ORF subsequent to the Flp-In™ reaction, even though the lacZ-Zeocin ORF does not have a bonafide promoter and ATG. In such cases, the hygromycin-resistant clones would also be lacZ-positive and Zeocin™ antibiotic-resistant. To make sure that the integration is FRT site-specific and not random, we recommend doing a parallel control transfection with no pOG44 present. This should yield no surviving clones upon hygromycin selection, indicating that all the hygromycin-resistant clones obtained in the presence of pOG44 are indeed Flp recombinase–dependent and hence have the gene of interest integrated at the FRT site. Also, a Southern blot analysis of these clones will help verify that they do indeed have proper FRT integration of the gene of interest despite the expression of lacZ (although this is typically not necessary). After the Flp-In™ reaction, as long as you see hygromycin-resistant clones, we recommend that you select them and assay them for expression of your gene of interest. 

Flp-In™ Jurkat cells are a little tricky to handle and are very sensitive to centrifugation. You may try the following protocol to thaw the cells:

  • Thaw 1 vial of cells into a T25 flask containing 5–7 mL of fresh medium (without selection). Do not spin down the cells to remove the DMSO at this point, as they are very sensitive to handling (including centrifugation). Typically, there is a lot of debris present upon thaw.
  • 24 hours post-thaw, spin down the cells at 900 rpm for 2–3 minutes and resuspend gently into 5 mL of fresh medium. Spinning down the entire volume of cells greatly reduces the amount of debris in the culture.
  • At day 5 or 6, it is okay to add the selection. Continue to passage the cells (spinning the cells down) every 2–3 days for a total of 1.5 weeks, each time resuspending back into only 5–7 ml of fresh medium in a T25 flask to build up the cell density. The Jurkat cells are very small and clumpy, and they expand very slowly.
  • Attempt to expand the cells into a T75 flask only after about 1.5 weeks.

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