Using 2A “self-cleaving” peptide in bicistronic mammalian expression

Multiple promoters or internal ribosomal entry sites (IRES) have been used for the production of multiple proteins from the same vector. Potential drawbacks with multiple promoters on viral vectors include unstable genome and interference between promoters. IRES is a relatively large sequence that can cause problems in virus packaging, especially for viruses with very limited genome size such as AAV. In addition, it is required that the start of the second ORF is fairly close to the IRES, adding difficulties to cloning.

2A or 2A-like peptide (collectively called 2A peptide here) is used by several families of viruses, the best known foot-and-mouth disease virus of the Picornaviridae family, for producing multiple polypeptides. Although called a “self-cleaving” peptide or protease site, the mechanism by the 2A sequence for generating two proteins from one transcript is by ribosome skipping–a normal peptide bond is impaired at 2A, resulting in two discontinuous protein fragments from one translation even.

The 2A-based bicistronic expression has been used for several years, but recently gained much more popularity due to its successful use in iPSC generation that required 2 to 4 factors working in concert. Even expression of all factors can be achieved when 2A peptides are used for multiple protein production, due to near 100% efficiency of the 2A “cleavage” at each site, and no interference between multiple 2A sites. Early work used a 36 amino acid sequence as 2A peptide, which was later reduced to about half that size from mutation and screening. Commercial vectors utilizing 2A for co-expression of cDNA and fluorescent protein and/or drug resistance genes have not been available until now. Allele Biotech has introduced a number of such plasmids, establishing another First-to-the-market as it has done many times previously in its 10 year history.

New product of the week 03-29-10 to 04-04-10:

Alleleustrious pmTFP1-2A Bicistronic mammalian expression vector, ABP-FP-T2A10, $399, http://www.allelebiotech.com/shopcart/index.php?c=215&sc=34

Promotion of the week 03-29-10 to 04-04-10:

Buy any GFP-Trap beads or kits, get polyclonal anti-GFP (ABP-PAB-PAGFP10) at half size for FREE!  ***GFP-Trap has been replaced with GFP-nAb, an improved version for the same purposes in the form of agarose beads, magnetic beads, polyacrylamide beads, spin column kits, etc.  Come and take a look at the most comprehensive list of nearly all FPs before purchasing.

Tags: 2A peptide, anti-GFP, bicistronic, drug resistance, GFP-Trap, iPS, iPSCs, IRES, polycistronic, ribosome skip, self-cleaving, transcription, translation

Lentivirus-lanYFP Give Away

Promotion of the week 03-22-10 to 03-28-10: To help researchers get familiar with pre-packaged lentivirus, we offer free high titer lentivirus carrying a truly bright and fast maturing lanYFP (lancelet FP, new exclusively from Allele).  Infect virtually any mammalian cells by a single manipulation (pipeting) and watch cells turn green/yellow in about a day under microscope or on FACS.  Primarily a yellow FP, lanYFP will show brighter fluorescence than EGFP even when observed using standard GFP/FITC filter set.

New product of the week 03-22-10 to 03-28-10: anti-mTFP1/mWasabi polyclonal antibodies.  It is tailored-made for Alleleustrious mTFP1 and mWasabi, the brightest teal and green FPs.

Tags: EGFP, Fluorescent proteins, GFP, Lancelet, lentivirus, mTFP1, mWasabi, pre-validated virus, YFP

Fluorescent Protein-Based Assay Development II

FPs as pH and redox sensors:

The uses of FPs extend well beyond simple expression and fusion reporters.  While pH sensitivity (usually quenching of fluorescence by acidic pH) is generally considered a drawback for fusion tagging, it becomes a useful property for constructing pH sensors.  FPs specifically engineered to take advantage of pH sensitivity (“pHluorins”) report pH as either a change in fluorescent intensity or a change in the ratio of excitation at two different wavelengths, and may be used to monitor processes such as endocytosis or other pH-variable processes.  In such an application, the pH-sensitive FP is fused to a localization tag for the compartment of interest which experiences variable pH.  This technique can be used, for example, to visualize release of neurotransmitter-containing vesicles.  In addition to pH-sensitive FPs, redox-sensitive Aequorea GFP variants have been produced (roGFPs and others) which produce similar changes in fluorescence intensity or excitation ratio when exposed to differing redox conditions or reactive oxygen species.

Sensors based on circularly permuted FPs:

Because FPs have such a compact and stable beta-barrel fold with N and C termini close together, it is possible to engineer circularly permuted variants which retain their fluorescent properties.  Studies on circular permutation of FPs have led to the development of several different sensors which take advantage of domains inserted into sensitive areas of the fluorescent protein backbone.  The most famous of these are the GCaMP calcium sensors, in which a calmodulin domain has been inserted into a loop in GFP, yielding a sensor that reports calcium concentration as a change in fluorescent intensity.  Other circularly permuted FP variants, such as cpVenus (a yellow Aequorea GFP variant), have found usefulness in improving FRET sensor dynamic range (see next section).

FRET sensors:

Fluorescence resonance energy transfer (FRET) is a quantum mechanical process that allows the transfer of excited state energy between two fluorophores when they are in close physical proximity.  Because this process operates with a strong distance (1/r^6) and orientation dependence (strongest when chromophore dipoles are parallel or antiparallel), it lends itself to the construction of highly sensitive reporters of biochemical activity.  In FP FRET, excited state energy from a higher-energy (shorter wavelength) “donor” fluorescent protein is transferred to a lower-energy (longer wavelength) “acceptor” FP, leading to sensitized fluorescent emission from the acceptor and reduced emission (quenching) from the donor.  By linking donor and acceptor FPs with a domain which changes conformation in response to a biochemical activity of interest, this activity is reported as a change in the ratio of sensitized emission to direct-excitation emission of the acceptor (or a simple ratio of donor and acceptor emission).  FRET sensors have been engineered to specifically sense a wide variety of activities, including many protein kinases, as well as small molecules such as Ca2+ and neurotransmitters.  While design of a new FRET sensor generally requires a great deal of optimization and trail-and-error, this class of probe is among the most powerful tools currently available for investigating live-cell biochemistry.

New Product of the Week 03-15-10 to 03-21-10: Oct4-Sox2 2-in-1 lentivirus ABP-SC-LVI2in1 for effective iPS generation link: http://www.allelebiotech.com/shopcart/index.php?c=132&sc=122.
Promotion of the Week 03-15-10 to 03-21-10: 5% off plate oligos at all scales! www.allelebiotech.com/allele3/Oligo_96Plate.php We are doing our “window promotion” again, during a hour-long window, get any Allele’s High efficiency competent cells at 30% regular price, the time will be announced tomorrow on our Facebook page.

Tags: animal models, biosensors, calmodulin, cell based assays, circular permutatioon, Fluorescent proteins, FP sensor, FRET, GCaMP, pH sensors, redox sensor

Fluorescent Protein-Based Assay Development

This blog is a preview of what is to be launched as a new Service Group. Allele Biotech is restructuring its CRO capabilities in the assay development area by combining its fast expanding fluorescent protein portfolio, viral vector and packaging expertise, as well as newly granted patents in shRNA. The focus of this post is fluorescent protein in biosensor and screening assays. A modified version will be used as the landing page for the FB-Based Assay Development Service.

Overview:

Originally cloned from the jellyfish Aequorea victoria and subsequently from many other marine organisms, fluorescent proteins (FPs) spanning the entire visual spectrum have become some of the most widely used genetically encoded tags. Unlike traditional labeling methods, FPs may be used to specifically label virtually any protein of interest in a living cell with minimal perturbation to its endogenous function. Genes encoding FPs alone or as fusions to a protein of interest may be introduced to cells by a number of different methods, including simple plasmid transfection or viral transduction. Once expressed, FPs are easily detected with standard fluorescence microscopy equipment.

Factors that should be taken into account when designing an FP-based imaging experiment include the desired wavelength(s) for detection, the pH environment of the tagged protein, the total required imaging time, and the expression level or dynamic range required for detection of promoter activity or tagged protein. Individual FPs currently available to the research community vary considerably in their photostability, pH sensitivity, and overall brightness, and so FPs must be chosen with care to maximize the likelihood of success in a particular experimental context.

FPs as fusion tags:

Use of FPs as fusion tags allows visualization of the dynamic localization of the tagged protein in living cells. For such applications, the cDNA of a protein of interest is attached in-frame to the coding sequence for the desired FP, and both are put under the control of a promoter appropriate to the experimental context (typically CMV for high-level expression, though other promoters may be desirable if overexpression of your protein of interest is suspected of producing artifacts). The most basic uses for fluorescent protein fusions include tracking of specific organelles (fusions to short organelle targeting signals) or cytoskeletal structures (fusions to actin or tubulin, for example). More advanced uses include tracking receptors or exported proteins. In most cases, it is critical that the FP used for fusion tagging be fully monomeric, as any interaction between fusion tags is likely to produce artifacts, some of which may be hard to recognize in the absence of other controls. While in most cases FP fusions do not interfere with normal protein function, whenever possible, FP fusion proteins should be validated by immunostaining the corresponding endogenous protein in non-transfected cells and verifying similar patterns of localization.

FPs as expression reporters:

FPs are highly useful as quantitative expression reporters. By driving the expression of an FP gene by a specific promoter of interest, it is possible to produce an optical readout of promoter activity. Use of the brightest possible FP ensures the best dynamic range for such an experiment. Because dynamic localization is not generally an issue for expression reporter applications, it is possible to use non-monomeric FPs for this purpose, opening up additional possibilities for multiple wavelength imaging. In order to obtain more reliable quantitative data and to correct for likely variations between individual cells in expression reporter experiments, the use of two spectrally distinct (e.g. green and red) FPs is advisable. By driving expression of one FP with a constitutive promoter and a second FP with the promoter of interest, the ratio of the two signals provides a quantitative readout of relative activity. Averaged over many cells, this technique should provide statistical power necessary for quality expression level experiments. Because FPs normally have a very slow turnover rate in mammalian cells, it may be desirable to add a degradation tag to your FP to enhance temporal resolution when measuring highly dynamic promoter activity.

New Product of the Week 03-08-10 to 03-14-10: mWasabi 2A or IRES dual expression vectors (http://www.allelebiotech.com/shopcart/index.php?c=216&sc=34) ABP-FP-W2A10, orWIRES10

Promotion of the Week 03-08-10 to 03-14-10: for a limited time on Thursday, to be announced on our Facebook page (http://www.facebook.com/pages/San-Diego-CA/Allele-Biotechnology-and-Pharmaceuticals-Inc/78331924957#!/allele.biotech?ref=profile), a strikingly low price will be honored for a commonly used lab reagent or equipment. This is the second week of the follow-us-to-the-basement promotion.

Tags: assay development, biosensor, cell based assays, chemical screening, circular permutation, fluorescent protein, FRET, GFP, mTFP1, mWasabi, promoter-FP, RNAi screening

How to Generate Conditional Knockout Mice with Cre

The bacterial Cre recombinase targets a specific DNA sequence called loxP and deletes a segment of DNA flanked by loxP sequences. This system is often used in the generation of knockout and conditional knockout animals.

The knockout of specific genes leading to embryonic lethal phenotype will not yield adult animals. Cre-lox recombination provides a means to knockout the specific genes in adult mice, or to introduce a knockout phenotype in specific tissues (conditional knockout) using tissue-specific promoter driven Cre or an inducible Cre.

The cutting by Cre at the loxP sites and rejoining by ligase is an efficient process. During this process, inverted loxP sites will result in an inversion, whereas direct repeat will cause a deletion. Cre/lox recombination is a one-way reaction so there is no need for continued Cre expression. Therefore, Cre can be introduced by adenovirus or lenti/retrovirus. Here is an example of using adnovirus-Cre in one lab: for MEF, on a 70% confluent P10 cm plate (probably 2-2.5 million cells), use 6ul of 1.1×10^12 adenovirus-Cre, which will give 80% infection; or use 10ul of 1.1×10^12 adenovirus-Cre to get 90% infection, with GFP as marker and analyzed by FACS.

Adenovirus could post a toxicity problem when used at very high titers to reach high percentage of transduction. An alternative is to use only lentivirus-Cre, at only about 1-2 ul and still obtain >80% infection. However, a silencing event needs to occur before the expression of Cre from lentivirus is shut off. The timing and degree of silencing is not controlled in such experiments. Continued expression of Cre should not influence most experiments.

To be certain that the Cre enzyme can be successfully delivered into the nucleus for conditional knockout to occur, the bacterial Cre gene needs to be engineered to contain a nuclear localization (nl) signal of eukaryotic cells. The function of the nuclear-localized Cre (nlCre) can be tested using a loxP-nuclear localized lacZ (nlacZ) reporter cell line, which can be used to monitor the function of the nlCre recombinase.

New Product of the Week 03-01-10 to 03-07-10: Lentivirus-Cre ABP-RP-TLCCreS and ABP-RP-TLCCreL, link: http://www.allelebiotech.com/shopcart/index.php?c=219&sc=188.

Promotion of the Week 03-01-10 to 03-07-10: for a limited time, get lab agarose at ~1/5 of the big company’s price. http://www.facebook.com/#!/pages/San-Diego-CA/Allele-Biotechnology-and-Pharmaceuticals-Inc/78331924957

Tags: adenovirus cre, conditional knockout, cre, cre loxp, cre recombinase, knockout mice, lentivirus cre, MEF