Basic Mammalian Expression Support—Getting Started

Even though mammalian expression is more time consuming and not as easy to use as expression in some other host systems, and also not as cost-effective, it is the system of choice for studying the function of a particular protein in the most physiologically relevant environment, since it allows for the highest level of post-translational processing of the protein.  

The table below lists the pros and cons of mammalian expression in comparison with expression in other host systems:

A dose-response curve or kill curve is a simple method for determining the optimal antibiotic concentration to use when establishing a stable cell line. Untransfected cells are grown in medium containing antibiotic at varying concentrations in order to determine the least amount of antibiotic needed to achieve complete cell death. The basic steps for performing a dose-response curve or kill curve are as follows:

• Plate untransfected cells at 25% confluence and grow them in medium containing increasing concentrations of the antibiotic. For some antibiotics, you will need to calculate the amount of active drug to control for lot variation.
• Replenish the selective medium every 3–4 days. After 10–12 days, examine the dishes for viable cells. The cells may divide once or twice in the selective medium before cell death begins to occur.
• Look for the minimum concentration of antibiotic that resulted in complete cell death. This is the optimal antibiotic concentration to use for stable selection.

Eukaryotic transcriptional termination signals are ill-defined. In our mammalian expression vectors, transcriptional termination is provided by the SV40 polyA, BGH polyA, or TK polyA site downstream of the multiple cloning site.

Eukaryotic (and specifically mammalian) mRNA contains sequence information that is important for efficient translation. This sequence, termed a Kozak sequence, is a translation initiation enhancer. The consensus Kozak sequence is ACCAUGG, where AUG is the initiation codon. Point mutations in the nucleotides surrounding the initiation codon have been shown to modulate translation efficiency. A purine (A/G) in position -3 has a dominant effect; with a pyrimidine (C/T) in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Expression levels can be reduced up to 95% when the -3 position is changed from a purine to pyrimidine. The +4 position has less influence on expression levels where approximately 50% reduction is seen. See the following references:

• Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292.
• Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947-950.
• Kozak, M. (1987) An analysis of 5´-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125-8148.
• Kozak, M. (1989) The scanning model for translation: An update. J. Cell Biol. 108, 229-241.
• Kozak, M. (1990) Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucleic Acids Res. 18, 2828.

The Kozak sequence is a translation initiation enhancer sequence present in eukaryotic (specifically mammalian) mRNA. The Kozak sequence is important for efficient translation. The consensus Kozak sequence is ACCAUGG, where AUG is the initiation codon. Point mutations in the nucleotides surrounding the initiation codon have been shown to modulate translation efficiency. A purine (A/G) in position -3 has a dominant effect; with a pyrimidine (C/T) in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Expression levels can be reduced up to 95% when the -3 position is changed from a purine to pyrimidine. The +4 position has less influence on expression levels where approximately 50% reduction is seen. See the following references:

• Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292.
• Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947-950.
• Kozak, M. (1987) An analysis of 5´-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125-8148.
• Kozak, M. (1989) The scanning model for translation: An update. J. Cell Biol. 108, 229-241.
• Kozak, M. (1990) Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucleic Acids Res. 18, 2828.

Although we make a general recommendation to include a Kozak consensus sequence, the necessity depends on the gene of interest and often, the ATG alone may be sufficient for efficient translation initiation. The best advice is to keep the native start site found in the cDNA unless one knows that it is not functionally ideal. If concerned about expression, it is advisable to test two constructs, one with the native start site and the other with a consensus Kozak sequence. In general, all expression vectors that have an N-terminal fusion will already have an initiation site for translation.

We do not offer a mammalian expression vector with a cleavable C-terminal tag.  

All of our antibiotics (Geneticin®, Zeocin™, Hygromycin B, Blasticidin, and Puromycin) can be used together for making multiple stable cell lines. However, kill curves will need to be performed for each combination of antibiotics since sensitivity to a given antibiotic tends to increase when combined with other antibiotics. 

The differences between the A, B, and C forms of our vectors are a result of either single base-pair addition or deletion in the multiple cloning site (MCS) of the vector. As a result of these single base changes, we have generated three separate reading frames for each type of vector. This feature will enable cloning a gene of interest in frame with the epitope tag using the restriction enzyme of choice. The three reading frames A, B, and C are provided in three separate tubes. 

The CMV promoter is known to be downregulated over time in mouse cell lines. Hence, we recommend using one of our non-CMV vectors, such as those with the EF1α or UbC promoter, for long-term expression in mouse cell lines.

We recommend using One Shot® ccdB Survival™ 2 T1^R Competent Cells, Cat. No. A10460. This strain is resistant to the toxic effects of the ccdB gene.

Note: Do notuse general E. coli cloning strains, including TOP10 or DH5α™, for propagation and maintenance, as these strains are sensitive to ccdB effects.

We offer pJTI™ R4 Exp CMV EmGFP pA Vector, Cat. No. A14146, which you can use to monitor your transfection and expression.

pcDNA3 is no longer available from Thermo Fisher Scientific but has been directly replaced by pcDNA3.1, which was derived from pcDNA3. The center of the multiple cloning site (MCS) within the original pcDNA3 vector contained homology to a hairpin mRNA structure and involved the Eag I, Not I, and both BstXI sequences. This hairpin would only have affected expression of genes cloned downstream of the Not I site, if at all. To address this issue, some sequences were removed, including the Eag I site, and the BstXI sequences were slightly modified to reduce homology. A 32-base fragment from pcDNA3 (between bases 995 and 1026), which contains the Sp6 primer site, was also removed and 11 bases were inserted in its place, adding another PmeI restriction site into the MCS of pcDNA3.1.

Here are the links to the vector sequences for pcDNA3, pcDNA3.1(+), and pcDNA3.1(−).

The (+) and (-) designations refer to the orientation of the multiple cloning sites in these vectors.  The availability of the cloning site in two orientations facilitates flexibility in cloning scheme design, so that if, for example, your insert must clone in as a Not I to Bam HI orientation, you may choose the (-) version of these vectors. 

pcDNA™3.1 vectors contain the core CMV promoter that is truncated before the start of transcription, whereas the pcDNA™ 3.3-TOPO® vector has the 672 bp native CMV promoter. This native CMV promoter allows high-level gene expression with two- to five-fold higher protein yields compared to other expression vectors. pcDNA™3.1 vectors are available in restriction, TOPO®, and Gateway® cloning versions and as untagged and epitope-tagged versions, whereas the pcDNA™3.3-TOPO® vector is a TOPO® TA-adapted, untagged vector that can be used to express native proteins without extraneous amino acids, and is hence ideal for antibody production and structural biology.

ThepcDNA™4/HisMax and pcDNA™4/HisMax-TOPO® vectors contain the QBI SP163 translational enhancer to increase expression levels two- to five-fold above those seen with the CMV promoter alone.

We offer three unique mammalian expression systems for inducible/regulated expression of the gene of interest:

• T-REx™ system
• Flp-In™ T-REx™ system
• GeneSwitch™ system

Please see the table below to see how they compare with one another:

We offer T-REx™-293, -HeLa, -CHO, and -Jurkat cell lines. These cell lines are derived by transfection of parental cells with pcDNA™6/TR followed by stable selection with blasticidin. They constitutively and stably express the TetR gene, allowing significant time and effort saving when using the T-REx™ system. These cell lines are functionally tested for expression by transient transfection with the positive control vector, pcDNA™4/TO/lacZ. They exhibit extremely low basal expression levels of bGal in the repressed state and high expression upon induction with tetracycline.

The Gateway® pT-REx™ DEST31 vector contains an N-terminal 6xHis tag (the 6XHis in this vector is not followed by Glycine (G) or -COOH). Hence, it cannot be detected using anti-HisG or anti-His (C-terminal) antibodies. Instead, we recommend using an anti-6xHis antibody (Cat. No. 372900).

We do not offer an anti-TetR antibody. Even though a western using an anti-TetR antibody can be used to screen out clones that do not express any TetR protein, it would not be the optimal way to screen for functional clones. Functional testing by performing a transient transfection with the lacZ expression control plasmid is recommended for this purpose, followed by picking a clone that shows lowest basal levels of expression of β-galactosidase in the absence of tetracycline, and highest levels of β-galactosidase expression upon addition of tetracycline.

Doxycycline may be used as an alternative inducing agent in the T-REx™ system. It is similar to tetracycline in its mechanism of action, and exhibits similar dose-response and induction characteristics as tetracycline in the T-REx™ system. Doxycycline has been shown to have a longer half-life than tetracycline (48 hours vs. 24 hours, respectively). We do not offer doxycycline, but it may be obtained from Sigma (Cat. No. D9891). 

The Flp-In™ T-REx™ system combines the targeted integration offered by the Flp-In™ system with the powerful inducible expression offered by the T-REx™ system. It allows generation of isogenic, inducible, stable cell lines and permits polyclonal selection of these cell lines. Once the Flp-In™ T-REx™ host cell line containing an integrated FRT site has been created, subsequent generation of Flp-In™ T-REx™ cell lines expressing the gene(s) of interest is rapid and efficient.

We offer the Flp-In™ T-REx™ system that contains pFRT/lacZeo and pcDNA6/TR stably integrated into HEK 293 cells. This cell line has been functionally tested for its ability to regulate expression.

With the GeneSwitch™ system, it is possible to have the absolute lowest basal levels of expression of the gene of interest, whereas the T-REx™ system may be a little leaky due to the inevitable presence of tetracycline in FBS. The induced level of expression in the GeneSwitch™ system can be even higher than that seen with the CMV promoter. The disadvantage of the GeneSwitch™ system is that the expression does not appear to switch off very easily in culture, although it has been demonstrated to function beautifully in transgenics. The T-REx™ system, on the other hand, can be switched on and off by the addition and removal of the inducer.

When a co-transfection is performed, there is no way of testing the double stable cell line for functional TetR or GeneSwitch™ protein, respectively. On the other hand, when sequential transfection is performed, one can functionally test the generated T-REx™ or GeneSwitch™ cell line by transiently transfecting the lacZ expression control plasmid and then picking a clone that shows the lowest basal level of expression of lacZ in the absence of the inducer, and the highest level of lacZ in the presence of the inducer. This clone can then be expanded and used to transfect the T-REx™ or GeneSwitch™ expression construct, as the case may be.

The GeneSwitch™ protein contains functional domains from different transcription factors, allowing it to function as a ligand-dependent transcription factor to activate expression of both the gene of interest and its own gene. The GeneSwitch™ protein exhibits the following characteristics:

• Since the GAL4 DBD is derived from a yeast protein, the GeneSwitch™ protein has no effect on endogenous genes and can only activate transcription of genes whose expression is controlled by a GAL4 UAS (i.e., the gene of interest and the regulatory fusion gene).
• The GAL4 DBD binds to an individual 17-nucleotide GAL4-binding site as a homodimer. The pGene/V5-His and pSwitch plasmids contain 6 and 4 copies of the GAL4 binding site, respectively, although it is not known if all of the GAL4-binding sites are occupied at any given time.
• The truncated hPR-LBD contains a 19 amino acid deletion from its C-terminal end that abolishes its ability to bind to progesterone, other endogenous steroid hormones, or other progesterone agonists, but still enables it to bind with high affinity to mifepristone.
• The p65 AD is a strong transcriptional activator but is derived from a human protein, to minimize possible toxic or pleiotropic effects associated with viral transactivation domains.

Sorry, all GeneSwitch™ cell lines have been discontinued.

The Ecdysone vectors and cell lines have been discontinued, but we do still offer the inducers, Muristerone A and Ponasterone A.

In theory, one can get multiple integrations of the Flp-In™ expression construct—an FRT-specific integration event and a random, second-site integration. However, random integration is a relatively uncommon event. Limiting the amount of DNA in the transfection will reduce the chance of second-site integration. We have transfected 293 cells (lacking the FRT site) with the pcDNA™5/FRT vector and have identified one potential second-site integrant after screening over 200 clones. DNA integrations can be detected by Southern blot. A single integrant will display a single band; double: two; triple: three, etc. We have maintained a number of Flp-In™ expression cell lines for over four months and have not observed any loss of the Flp-In™ expression construct, whether hygromycin selection was maintained or not.

The Jump-In™ system is PhiC31-integrase mediated and is a stable, targeted, and irreversible mammalian expression system. It consists of the Jump-In™ Fast system that involves a single integration step and the Jump-In™TI™ (targeted integration) system that needs two integration steps, both of which are targeted and irreversible. In contrast, the Flp-In™ system is a stable, targeted mammalian expression system that is reversible. The first integration is random (integration of pFRT/lacZeo), and the second integration (integration of the Flp-In™ expression vector) is targeted but reversible.

We recommend using the Jump-In™ Fast system if you need stable mammalian expression and want to quickly generate well-expressing clones. You can have well-expressing clones with one or more integrations at the PhiC31 pseudo-att P sites. A Southern blot is necessary to confirm the number of integrated events. Use the Jump-In™TI™ system if you need isogenic expression, where every cloned gene would be expressed from the same locus in the same background, with no chromosomal position effects.

The amount of DNA to be used to obtain single copies should be determined by control experiments done in the absence of integrase. The same amount of DNA that yields less than 5 colonies in the absence of integrase should be used in the presence of integrase. Typically, the integrase expression plasmid makes up most of the amount of DNA used for transfection.

A platform cell line is created when the R4 attP retargeting sequences are site-specifically inserted into the mammalian genome via PhiC31 Int-mediated recombination. In addition to the R4 retargeting sequences, this integration event introduces the hygromycin resistance gene under the control of the HSV TK promoter, and the promoterless Bsd, Neo, or Zeo resistance marker, depending on the platform vector used (i.e., pJTI™/Bsd, pJTI™/Neo, or pJTI™/Zeo). Although you select for transformants carrying the R4 retargeting sequences by their resistance to hygromycin, you may perform PCR analysis to check the integrity of the R4 attP retargeting sequences. For this, we recommend amplifying the region from the R4 attP sequence to the appropriate resistance marker (depending on the platform line used) using the genomic DNA from the platform line. A nested PCR is recommended to reduce the high background you may observe with only primary PCR. Alternatively, you may create a labeled DNA probe by PCR amplifying an approximately 1.5 kb region covering the retargeting sequences, and then perform a Southern blot analysis. The Southern blot will also act as an additional check to verify that only a single copy of the retargeting sequence is integrated into the genome.

The second step in targeted integration is the retargeting event mediated by the R4 integrase, where the genetic elements of interest are site-specifically transferred from the retargeting expression construct (created using the MultiSite Gateway® Pro module) into the genome of the platform line. This integration event also positions the EF1α promoter upstream of the blasticidin, neomycin, or Zeocin™ resistance gene (i.e., “promoterless” selection marker), thus allowing the selection of transformants that are successfully “retargeted” using the appropriate selection agent. Although you select from successfully retargeted clones using blasticidin, Geneticin®, or Zeocin™ antibiotic, you may also perform a nested PCR to amplify the region from the EF1α promoter to the appropriate resistance gene. You can amplify the hygromycin resistance gene as a positive control. Similar to the platform line creation, you may also perform a Southern blot analysis with a probe designed for your gene of interest.

The pJTI™ Phic31 Int vector does not contain an NLS. Adding an NLS could increase the efficiency of site-specific integration at pseudo attP sites, but there are no data to support it. There is one paper describing the use of an NLS on a PhiC31 integrase vector, but the authors didn't measure integration into pseudo attP sites.

We would recommend engineering an expression marker/reporter in the plasmid used to create the platform line, and then screening the platform line for expression of this marker to identify a high-expressing locus. Otherwise, the process can get quite labor-intensive, as multiple lines would have to be screened after retargeting.