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

Monitoring the Undifferentiated Stage of Stem Cells—the Pluripotency Markers

Human embryonic stem (ES) cells or induced pluripotent stem (iPS) cells promise to serve as an unlimited source for transplantation or tissue-specific differentiation. However, obtaining and maintaining stem cells are very difficult tasks for multiple reasons. For instance, most stem cell lines tend to spontaneously differentiate in culture, and even if the cells form stem cell-like colonies, they may be of a heterogeneous population.

To identify pluripotency of stem cells, expression of stem cell-specific marker genes (i.e. Oct-3/4, Sox2, Nanog, Rex-1) is monitored by RT-PCR. Alkaline phosphatase activity and methylation profiles of promoters of pluripotency-relevant genes are often analyzed as well. Compared to murine cells, it is noticeably more difficult to obtain human iPSCs, of which stem cell-like colonies sometimes turn out not to be pluripotent cells. We highly recommend testing iPSCs, especially human iPSCs, with antibodies against stage-specific embryonic antigens such as SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81.

However, all of these methods require cell destruction or fixation for analysis, therefore, are inconvenient and costly. Furthermore, many studies using ES or iPS cells involve differentiation of stem cells into different lineages, a method for observing live cells to know their undifferentiation/differentiation stages would be very helpful. There have been a number of publications using murine Oct-4, Nanog, and Rex-1 promoter driven fluorescent proteins as markers for pluripotency tests [1-3]. Allele Biotech provides, under its iPS product line, packaged and validated lentiviral particles that would insert these 3 promoter-FP reporters into the stem cells. Although currently these promoters are of mouse sequences, their use in human stem cells have been reported.

New product of the week 01-25-10 to 01-31-10:

All-In-One-Vector: Human OSKM Lentiviral Paticles, with Oct-4, Sox-2, Klf, and c-Myc all expressed from a single virus, ready-to-use.

• Promotion of the week:

human iPS cell detection primer set, the same as the landmark Yamanaka paper [4] on creating human iPS for the first time.

1. Da Yong WU, Zhen YAO (2005). Isolation and characterization of the murine Nanog gene promoter. Cell Research, 15 (5): 317–324.
2. Rachel Eiges, Maya Schuldiner..et.al (2001). Establishment of human embryonic stem cell?transfected clones carrying a marker for undifferentiated cell. Current Biology 11: 514–518.
3. Guangjin Pan, Jun Li, Yali Zhou, Hui Zheng, and Duanqing pei (2006). A negative feedback loop of transcription factors that control stem cell pluripotency and self?renewal. ASEB Journal 20: E1094? E1102
4. Takahashi et al, Induction of Pluripotent Stem Cell from Adult Human Fibroblasts by Defined Factors (2007). Cell 131, 861-872

Tags: differentiation, fluorescent protein, iPS, iPS lentivirus, iPSC, iPSCs, lentivirus, pluripotency reporter, promoter, promoter-FP, stem cells, undifferentiation stage

Allele Pre-packaged, Titer-determined, Validated iPS Generating Lentivirus Particles

Induced pluripotent stem cell (iPSC) has been an important research area in the past 3 years. These cells have also provided unprecedented possibilities to study cell differentiation and tissue development to biologists in many fields. Allele pre-packaged iPS lentiviral particles are validated, ready-to-use, high quality reagents for any laboratory to create iPS cells. A straight-forward and optimized protocol is also provided for immediate use of these products. iPC cells can be produced typically in 2-3 weeks; but when used in combination with shRNA against p53 (also available from Allele), iPSCs can be produced in just about 5 days!

Follow iPS news on our news page.

Tags: iPS, lentiviral particles, lentivirus, optimized protocol for inducing iPSC, Pre-packaged viral particles, quick iPS induction, titer-determined, validated iPSC

Why Allele?

Allele provides you with tools that you will find very helpful.  The two main motives for Allele developed products are:
1) To incorporate the most advanced technologies in the field
2) To provide equal utility as other companies’ equivalent products at a much more reasonable cost.

How did we do it?        By developing technologies internally, in most cases with government grant funding, by in-licensing others’ discoveries, and by listening to you, our customers.

What else do we do?      Conduct basic curiosity-driven research just like most of our customers. It helps to stay on the edge and connect to the community.

Tags: Allele, Allele Biotech, biomedical research, cost effectiveness, cost efficiency, DNA, Fluorescent proteins, iPS, low cost, Protein, reagents, RNAi, viruses

Allele Mail Bag: Our discovery that stem cells and other cells can be non-invasively intranasally delivered to the brain.

A few words from the blog editor:  The Allele Mail Bag is a new feature we initiated here by this post on July 20, 2009.  We like to post some of the messages that our research, business development, or customer service staff receives through Allele’s published email boxes, e.g. oligo@allelebiotech.com (for ordering any product or service), iPS@allelebiotech.com, RNAi@allelebiotech.com, FP@allelebiotech.com, Vivec@allelebiotech.com (for consulting with Allele experts on each of the focus product group. FP: fluorescent proteins.  Vivec: viral vectors).  If we find your message to be suitable as a guest post on our blog, we will ask for your permission first.

In addition to any questions about any product, service, or R&D activity that Allele may provide or perform, we also encourage you to use our communication and social networking channels to help more people become aware of your own research progress.  After all, it is by the same principle of scientific information exchange through traditional channels such as publication in journals or presentation at meetings—the better we communicate the more science benefits.

Excerpt from a recent email to iPS@allelebiotech.com, with permission from Dr. Frey:

“Hi,
I am excited to tell you that along with my collaborators in Germany, especially Lusine Danielyan MD, I have discovered that stem cells and other therapeutic cells can be non-invasively delivered to the brain using the intranasal delivery method that I developed.  The first of our papers on this new discovery was just published in European Journal of Cell Biology.  I have attached a copy of this paper.  I am hopeful that this    breakthrough, that could revolutionize the stem cell industry and make stem    cell treatments practical by eliminating the need for invasive neurosurgical implantation of cells, can facilitate the development of stem cell therapies for Parkinson’s disease, Alzheimer’s disease, stroke, traumatic brain injury and many other brain disorders.

Best Regards,
—
William H. Frey II, Ph.D., Director
Alzheimer’s Research Center
Regions Hospital
640 Jackson St.
St. Paul, MN 55101
Professor of Pharmaceutics, Neurology
and Neuroscience
University of Minnesota”

Tags: Alzheimer, brain research, cutting edge research, iPS, iPS retroviral gene delivery, lentiviral iPS vectors, neurosurgical implantation, new discovery, non-invasive delivery, Parkinson's disease, stem cells, stroke