Reporter & Specialty Vectors for Mammalian Expression Support—Getting Started

In addition to the key mutations that enhanced the brightness of the Clontech fluorescent proteins, we have added further genetic enhancements to the fluorescent proteins to increase the quantum yield. Side-by-side comparisons have shown the fluorescence intensity of our Vivid Colors™ fluorescent protein expression vectors to be at least equivalent (or better than) the comparable Clontech BD Living Colors™ fluorescent protein expression vectors. 

EmGFP is the next-generation variant of EGFP, and it has been further optimized for mammalian expression. Both EmGFP and EGFP can be visualized using the same filter sets (FITC) and settings. When used with the recommended filter sets and settings, Cycle 3 GFP is as bright as EGFP or EmGFP. However, when used with FITC filter sets and settings, Cycle 3 GFP is not as bright as EmGFP or EGFP.

Excitation/emission maxima for EGFP: 488 nm/507–509 nm
Excitation/emission maxima for EmGFP: 487 nm/509 nm
Excitation/emission maxima for Cycle 3 GFP: 395 nm (primary) and 478 nm (secondary)/507 nm 

Yes, all of the fluorescent proteins offered by us (EmGFP, YFP, CFP, BFP, and Cycle 3 GFP) have been humanized for optimal mammalian expression. 

Excitation and emission maxima for our fluorescent proteins are as follows:
EmGFP: Excitation: 487 nm; Emission: 509 nm
YFP: Excitation: 514 nm; Emission: 527 nm
BFP: Excitation: 308–383 nm; Emission: 440–447 nm
CFP: Excitation: 452 nm; Emission: 505 nm
Cycle 3 GFP: Primary excitation: 395 nm; Secondary Excitation: 478 nm; Emission: 507 nm

EmGFP, YFP, CFP, and BFP can be detected using standard FITC filter sets and settings. However, for optimal detection of the fluorescence signal, filter sets optimized for detection within the excitation and emission ranges for each fluorescent protein are recommended. The recommended filter sets are as follows:

EmGFP: Omega filter set XF100

YFP: Omega filter set XF1042
Chroma filter set 41028

CFP: Omega filter set XF114
Chroma filter set 31044

BFP: Omega filter set XF10
Chroma filter set 31021

For information on obtaining filter sets, please contact Omega Optical, Inc. (www.omegafilters.com) or Chroma Technology Corporation (www.chroma.com) directly. 

Cycle 3 GFP fluorescence can be detected using a filter set designed to detect wild-type GFP (since they have the same fluorescence spectra). In-house, we use the XF76 filter set from Omega Optical. For Cycle 3 GFP, excite at 395 nm and read emission at 507 nm. You can also look at the emission spectra and record emissions from 200–800 nm.

Cycle 3 GFP fluorescence can be quantitated with any type of fluorometer with the appropriate filters and cut-off wavelengths. In-house, we have a Hitachi F-2000 Fluorescence Spectrophotometer. Our general protocol using this machine is as follows:
Dilute samples in PBS (although Tris or water would be okay). The amount of lysate to be used will of course depend upon the concentration of GFP. This will have to be determined empirically. The primary consideration is that one needs to be in the linear range of the fluorometer. We have used quantities from 5–50 µL in 1 mL of PBS in a cuvette. If readings are going to be internally compared, the most consistent results will be obtained if the amounts of lysate used are normalized to either the transfection efficiency or the total protein concentration.

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

Yes, we do offer the pBlue-TOPO® and pGlow-TOPO® vectors that facilitate cloning of the DNA sequence of interest directly upstream of either the b-galactosidase or Cycle 3 GFP gene, respectively.   

pBlue-TOPO® is ideal for functional analysis of promoters with low transcriptional activity, since assays for β-galactosidase are easy to perform and are quantitative at very low levels of expression. pGlow-TOPO® is ideal for non-invasive analysis of promoter elements within intact, living cells. The fluorescent property of Cycle 3 GFP allows in vivo detection in virtually any cell type or species using microscopy with wild-type GFP filter sets or by fluorescence-activated cell sorting methods.

pBlue-TOPO® contains a cryptic prokaryotic promoter upstream of the lacZ reporter gene, due to which E. coli transformants may appear to be light blue when screened on plates containing X-Gal. Hence, we do not recommend using pBlue-TOPO® to evaluate promoter function in E. coli. However, pGlow-TOPO® can be used for these studies. Note that background expression of β-galactosidase from pBlue-TOPO® does not occur in mammalian cells.

The advantage of Lumio™ staining is that one can do both in vivo and in vitro protein labeling. For in vivo labeling, load the cells with the Lumio™ reagent and then visualize the cells/proteins under a fluorescence microscope. This is similar to the GeneBLAzer® detection procedure except that GeneBLAzer® detection is based on an enzymatic reaction that amplifies the reporter signal. GFP fluorescence can only be detected within the cell (in vivo) because proper protein folding is needed. The Lumio™ tag is very small (6 amino acids, 585 Da), in contrast to the bla protein in GeneBLAzer® detection (264 amino acids, 29 kDa) and the GFP protein (27 kDa), and therefore most likely will not interfere with the function of the protein it is fused to. GFP has the disadvantage of being a large fusion tag and is not an enzymatic-based reporter system.  Unlike GeneBLAzer® detection and GFP, a Lumio™-tagged protein can be visualized on a gel after treating the cell lysate or protein with the Lumio™ reagent. Compared to Lumio™ and GFP, GeneBLAzer® detection is a more sensitive detection method for use in live cells. Also unlike Lumio™ and GFP, the GeneBLAzer® detection method allows for ratiometric read-outs and thus eliminates sample-to-sample variation.

We have not experienced negative effects with Lumio™ reagents at the concentrations used to detect protein in the cells. We also do not see any change in cell morphology when using Lumio™ Green. After application of the Lumio™ Red, we do see some minor morphological changes in the cells that are reversed after 24 hours of application of the reagent. 

Serum proteins such as BSA (66 kDa) from the mammalian cell culture medium may cross-react with the Lumio™ reagent, producing non-specific bands. Removing the cell culture medium and washing the mammalian cells 3–4 times with PBS after harvesting the cells minimizes the non-specific binding from BSA. 

The Lumio™ reagent is hydrophobic and can easily pass through the membrane. There is no need to permeabilize the membrane in order to get this reagent into cells. 

You can accomplish this by designing a bicistronic transcript, in which the two genes are separated by an internal ribosome entry site (IRES) sequence and are expressed as a single transcriptional cassette under the control of a common upstream promoter. Alternatively, you can use a vector containing two separate promoters to drive expression of the two genes, thus maintaining the gene copy number within the cells. We offer the pBud/CE4.1 vector, Cat. No. V53220, designed for the independent expression of two genes from a single plasmid. It contains the CMV and EF1α promoters to provide almost equivalent expression of the two genes being expressed. It carries the Zeocin™ antibiotic-resistance marker for stable selection.

We carry the pVAX1 vector, Cat. No. V26020, engineered according to FDA guidelines, for this purpose. In this vector, eukaryotic DNA sequences are limited to those required for expression in order to minimize the possibility of chromosomal integration. It has the kanamycin resistance marker for selection in E. coli instead of ampicillin, which has been reported to cause an allergic response in some individuals.

We offer the pDisplay™ vector, Cat. No. V66020, designed to target recombinant proteins to the surface of mammalian cells. It contains an N-terminal murine Ig kappa-chain secretion signal and a C-terminal transmembrane anchoring domain from Platelet-derived Growth Factor Receptor (PDGFR) to target and anchor the protein of interest to the cell surface.

Transfection of pDisplay™ vector alone into mammalian cells does not result in a displayed polypeptide because the Ig kappa signal peptide and the PDGFR transmembrane domain are not in the same reading frame, and the ORF containing the kappa signal peptide ends 5 bp before the start of what is defined as the PDGFR transmembrane domain. 

Trypsin digests proteins at arginine or lysine residues, and it is widely accepted that the extracellular domains of membrane proteins are digested upon trypsinization of cells. But typically these proteins are rapidly replaced once the trypsin is removed. As an alternative to trypsin, you can remove most adherent cells using Versene (Cat. No. 15040066), which is a sterile 0.5 mM EDTA solution in PBS, and/or scraping. The EDTA chelates any free Mg²⁺ or Ca²⁺ ions, which are necessary for maintaining many cell attachments. Cell Dissociation Buffer, Enzyme-free, Hank’s-based (Cat. No. 13150016) or PBS-based (Cat. No. 13151014), which are cocktails of chelating agents, are also fine for this application and may be more effective than EDTA alone, and would be unlikely to adversely affect the receptor protein.

When we tried FACS cell sorting of pDisplay™-transfected cells using our anti-myc antibody, the sorting was not very efficient. However, pDisplay™-transfected cells can be isolated using magnetic beads by first incubating the cells with anti-myc antibody and then incubating the cell-antibody complex with magnetic beads that have anti-mouse IgG1 conjugated to them. The cell can then be isolated using a magnet.

You can insert an N-terminal secretion signal or leader sequence upstream of your gene and in-frame with the gene sequence to facilitate secreted expression of the protein. We actually offer the pSecTag2 (Cat. No. V90020) and pSecTag2/Hygro (Cat. No. V91020) vectors designed for this purpose. These vectors contain the N-terminal murine Ig kappa-chain secretion signal for secreted expression of the protein of interest.

The only difference between these vectors is that the pSecTag2 vectors have the Zeocin™ antibiotic-resistance gene for stable selection, whereas the pSecTag2/Hygro vectors have the hygromycin B resistance gene for stable selection.

Yes, you can use Zeocin™ antibiotic for selection in E. coli. However, keep in mind that for Zeocin™ antibiotic to be active, the salt concentration of the medium must remain low (<90 mM) and the pH must be 7.5. Prepare LB broth and LB agar plates using low-salt (5 g NaCl/liter) LB. 

pSectag2 vectors have the Zeocin™ antibiotic-resistance marker for selection in E. coli, and any E. coli strain that contains the complete Tn5 transposable element (i.e., DH5αF′IQ, SURE, SURE2) encodes the ble (bleomycin) resistance gene that confers resistance to the Zeocin™ antibiotic. Hence, for the most efficient selection, we highly recommend choosing an E. coli strain that does not contain the Tn5 gene.

The (+) and (-) designations refer to the orientation of the multiple cloning sites in these vectors. The vector is offered in two orientations to allow flexibility in cloning.

pShooter™ vectors are designed to localize recombinant proteins to intracellular compartments, such as the nucleus (pEF/myc/nuc and pCMV/myc/nuc), mitochondria (pCMV/myc/mito), endoplasmic reticulum (pCMV/myc/ER), or cytoplasm (pCMV/myc/cyto). 

The ER-targeting signal in pCMV/myc/ER vector consists of an N-terminal signal peptide to direct the protein into the secretory compartment and a C-terminal peptide (SEKDEL) to retain the protein in the ER. This results in the protein being retained in the ER lumen by binding to a receptor. The original reference for this vector is: Munro S and Pelham HRB (1987) A C-Terminal Signal Prevents Secretion of Luminal ER Proteins. Cell 48:899–907. 

In the pCMV/myc/mito vector, the mitochondrial targeting signal is an N-terminal signal sequence (presequence of the subunit VIII of human cytochrome C oxidase), which lets the fusion go through the mitochondrial membrane. The signal sequence is cleaved off so the fusion is inside the mitochondria (but also may be associated with the inner membrane).

Our episomal mammalian expression vectors (pCEP4, pREP4, and pEBNA-DEST) contain the Epstein Barr Virus (EBV) origin of replication (oriP) and the Epstein-Barr nuclear antigen (EBNA-1) for high-copy, transient, or stable episomal replication in human, primate, canine, and porcine cell lines. They do not bring about episomal expression in murine or rodent cell lines. pEBNA-DEST may also be used with stem cells.