Lentiviruses expressing constitutively active or dominant negative versions of genes in signaling pathways

NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) refers to a protein complex functional in signaling pathways, particularly in response to stress stimuli. There are two signaling pathways leading to the activation of NF-kB signaling, known as the canonical (or classical) pathway, the non-canonical (or alternative) pathway.

In the canonical NF-kB pathway, NF-kB dimers such as p50/RelA are maintained in the cytoplasm by interaction with an independent Inhibitor of NF-kB (IkB) molecule. When the upstream signaling is active, an IkBa kinase (IKK) complex consisting of catalytic kinase subunits IKKa and/or IKKb and the scaffold protein NEMO will be recruited to the cytoplasmic adaptor of certain cell surface receptor and stay activated. Activation of IKK complex will consequently phosphorylate the IkB at two serine residues, which induce the proteasomal degradation of IkB. Released from IkB, NF-kB dimers then translocate into the nucleus and bind with a consensus sequence (GGGACTTTCC) of various genes and thereby activates their transcription. In a negative feedback loop, NF-kB activation also leads to the expression of the IkB gene, which subsequently sequesters NF-kB subunits and terminates transcriptional activity unless a constitutive activation signal is present.

The non-canonical pathway is mainly for activation of p100/RelB complexes during B-and T-cell development, where NF-kB was first discovered. Different from the canonical pathway, 1) only certain receptor signals (e.g., Lymphotoxin B, B-cell activating factor, CD40) can activate this pathway, 2) it proceeds through an IKK complex that contains two IKKa subunits (but not NEMO) and 3) receptor binding leads to activation of the NF-kB-inducing kinase NIK, which phosphorylates and activates an IKKa complex, the latter in turn phosphorylates two serine residues adjacent to the ankyrin repeat C-terminal IkB domain of p100, leading to its partial proteolysis and liberation of the p52/RelB complex.

Other distinct NF-kB pathways undoubtedly exist. For example, p50 (or p52) homodimers enter the nucleus, where they become transcriptional activators by virtue of interaction with the IkB-like co-activator Bcl-3 (or IkBz). How these are regulated is not known.

There are many ways of applying reagents that can manipulate the NF-kB system for drug screening as well as basic research. For this reason, Allele Biotech is introducing a set of lentiviruses as listed below. The way this product group is operated is that the Allele will publish the design and specifics of the products, but will produce it upon the first order. The person who places the first order will need to wait for 3-5 weeks for receiving the products, but will receive a 40% discount and an opportunity to provide input to the product design. The platform for lentiviral products is based on our field-leading high titer lentivirus packaging capabilities, and you can rest assured that high quality lentivirus will be produced at a pace much faster and lower price than if you were to make them by your own lab or find them from anywhere else. Allele Biotech will introduce more such products in the fields of miRNA (to provide all ~800 human miRNA and their anti-miRNA silencers on virus), cell differentiation lineage-specific promoter-FP reporters, etc. We now call these products the Lead Products. Cost effectiveness is just the beginning. Imagine how our ingenuity and innovation can pave the way for your research.

1) HiTiter IkBa Expression Lentiviral Particles: can be used in functional studies of IkBa’s roles in NF-kB signaling. Overexpression of IkBa will eliminate the low-level activation by factors in the cell culture medium or other fluctuations to ensure that any change in NF-kB signaling is caused by the stimulus being tested.
2) HiTiter Dominant Negative IkBa Expression Lentiviral Particles: Dominant Negative IkBa has serine-to-alanine changes at residues 32 and 36. It can be used to repress the expression of endogenous IkBa or block NFkB signaling in certain cell lines.
3) HiTiter IkBa-RFP Fusion Expression Lentiviral Particles: This is a reporter suitable for the studying proteasomal degradation of IkBa after phosphorylation.

4) HiTiter IKKa Expression Lentiviral Particles: can be used in functional research for the role of IKKa in NF-kB signaling.
5) HiTiter Constitutively Active IKKa Expression Lentiviral Particles: Constitutively active IKKa has serine-to-glutamate mutations at residues 176 and 180.
6) HiTiter Dominant Negative IKKa Expression Lentiviral Particles: Dominant negative IKKa is mutated by serine-to-alanine at residues 176 and 180.
7) HiTiter IKKb Expression Lentiviral Particles: can be used in functional research for the role of IKKb in NF-kB signaling.
8) HiTiter Constitutively Active IKKb Expression Lentiviral Particles: Dominant negative IKKb is mutated by serine-to-glutamate at residues 177 and 181.
9) HiTiter Dominant Negative IKKb Expression Lentiviral Particles: Dominant negative IKKb is mutated by serine-to-alanine at residues 177 and 181.
10) HiTiter pNFkB-Luciferase Lentiviral Particles: a cis-reporter plasmid containing the luciferase reporter cDNA linked to five repeats of an NF-kB binding site.
11) HiTiter pNFkB-GFP Lentiviral Particles: a cis-reporter plasmid containing the GFP reporter gene linked to five repeats of an NF-kB binding site.

New Product of the Week 05-23-10 to 05-31-10:

Cre recombinase fused to mWasabi GFP carried on lentivirus, ABP-RP-Cre2AGL, or ABP-RP-Cre2AGS

Promotion of the week 05-23-10 to 05-31-10:

actually 3 promotions this week, first announced to Allele fans and friends on Facebook: 10% off the brand-new RFP-Trap ACT-CM-RFA0050 for co-IP with mCherry, mPlum, mOrange…Order by Monday to qualify for discount! Free sample for FAM-amidite for oligo cores at universities that make qPCR and other probes. Free sample of high attachment tissue culture plates that use 40% less plastics (made in Canada)

Tags: Bcl-3, drug screening, IkBa, IkBz, Ikka, Ikkb, lentivirus, NEMO, NF-kB, p100, p50/RelA, p52/RelB, signaling pathways, stimuli, stimulus

Telling Good iPSCs from Bad iPSCs

Since its discovery pluripotent stem cells (iPSCs) have been known to differ somewhat from embryonic stem cells (ESCs) in term of gene expression profiles. It also appears that only a small percentage of iPSCs have the full potential of stem cells defined by being able to develop into adult animals. Instead of a global pattern of variations, surprisingly, the difference between iPSC and ESC was found to localize in a small region of one chromosome in mouse, 12qF1, which could account for most iPS cells’ lack of complete pluripotency (Stadtfeld et al, Nature 2010). In this region resides an imprinted gene cluster that includes 2 non-coding genes, Gtl2 and Rian, that remain silenced in most iPSCs. The underlining mechanism is hypermethylation and hypoacetylation, resulting in “paternalizaition” of the region. The effects are manifested around the mid-gestation stage.

By adding histone deacetylase inhibitor valproic acid (VPA) the silenced gene cluster may be reactivated and the iPSCs so treated show increased Gtl2 expression and ability to give rise to normal embryos. Expression of other imprinted genes showed clone-to-clone variations, as was previously reported by a number of groups, but no consistent differences between ESCs cells and iPSCs. Therefore, by analyzing the expression levels of just two genes, Gtl2 and Rian, the potential of iPSCs to be fully pluripotent can be assessed.

The relationships between stem cell status and epigenetic repressions also include the recent finding that Oct4 and Sox2, which are both germ cell-specific and critical reprogramming factors, may be implicated in the regulation of Xist and Tsix RNAs that control epigenetic silencing of X chromosome in female embryos.

New Product of the Week 05-17-10 to 05-23-10: RT-PCR primer set, ABP-SC-iPSh4NX $49, for identifying exogenous iPS factor expression from 4-in-1 iPS lentivirus

Promotion of the Week 05-17-10 to 05-23-10: $85 off IceCube dry bath 0-75C variable temp

Tags: 12qF1, ES, ESC, Gtl2, Gtl2off, Gtl2on, imprinted, impriting, iPS, iPSC, pluripotency, Rian, silenced, VPA

BioTechniques Publishes Article on Single Domain Antibodies

Many blogs start by asking “Did you know…” to intrigue you to read along. So here it goes:

Did you know that there are more than 300,000 antibodies that are commercially available? And yes, many antibody companies are still generating more antibodies at ever faster pace and in a more systematic way. There are companies that plan to make peptide or short protein fragments for making antibodies against all human proteins or subproteome, others develop antibodies particularly suitable for demanding assays such as ChIP-CHIP. Government activities such as the National Cancer Institute (NCI)’s Clinical Proteomic Technologies Initiative (CPTI) and the Road Map program under the NIH Director’s Office also set goals of producing comprehensive sets of widely usable, renewable, affinity reagents for clinical cancer samples or the human proteome. Apparently people do not think the 300,000 available antibodies are sufficient for what they do.

Did you know that conventional antibodies commonly used as reagents are ~150kDa in molecular weight and can hardly be used inside live cells? Ulrich Rothbauer, professor in the department of biology at Ludwig Maximilians University, who is working with colleagues to develop tools to study cellular processes in living cells. “These antibodies have to assemble four different chains, two heavy and two light, and they’re assembled by disulfide bonds that cannot be correctly formed in the reducing environment of the cytoplasm. You cannot express such a huge complex molecule in living cells. You can [introduce] them by microinjection, for example, but it’s not applicable for high-throughput cell imaging.” [1] Antibody fragments such as scFv, Fab, and similar derivatives have been developed over the years to certain level of success, but not as widely accepted or practically amenable to replacing conventional antibodies.

Did you know that camel, llama, and shark naturally produce single heavy chain antibodies that can function as 13-16kDa fragments (yes if you have read previous Allele Blogs http://allelebiotech.com/blogs/2009/08/camelid-antibodies/)? They can easily be produced in bacteria, used directly inside live cells via transgene, fused to other proteins as a fusion tag, linked to DNA oligos as a detection module, or immobilized on beads for pull down or co-IP. Currently, these antibodies need to be selected by display after obtaining immunized antibody libraries. There is generally no commercial service for creating custom camelid antibodies at this time due to patent and other issues. Existing products are available for jelly fish GFP and DsRed derived RFP fusions. Publications using such a limited number of camelid antibodies have been amazing so far—dozens in top journals within the last few months and after only a short period of time since product launch.

New Product of the Week 05-09-10 to 05-16-10: RFP-Trap for mCherry, mRFP1, mOrange, mPlum, and mRuby etc.

Promotion of the Week 05-09-10 to 05-16-10: Purchase our ThermoExp500 PCR Thermocycer for $4,650.00, and qualify for $200 off or for a $300 credit toward any other Allele Biotech product or service! http://www.allelebiotech.com/allele3/EQ.php

Original BioTechniques Article http://www.biotechniques.com/news/biotechniquesNews/biotechniques-257771.html?utm_source=BioTechniques+Newsletters+%2526+e-Alerts&utm_campaign=b94f127de0-Methods+Newsletter&utm_medium=email

Tags: Alpaca, camelid antibodies, Chromotek, Fab, GFP-Trap, Hc antibodies, mCherry, mOrange, mPlum, mRFP1, mRuby, nAbs, RFP-Trap, scFv, single domain antibodies, VHH, VHH antibodies

Take Advantage of Allele’s Essential Virus Grant Program, Have High Titer Virus for Your Research on Essential Human Genes

The number of Allele products in the form of viral particles is increasing quickly. Most of these products are also listed under product groups categorized according to their respective functions (e.g. iPS production under “Induced Pluripotent Stem Cells”, shRNA under “RNA Interference”, cytokines and enzymes under “Recombinant Proteins”). Additional background information, as well as related products, may be found on the landing pages of each product group.

The increase in scientific publications reporting the conversion of “dormant” genes into “active” ones has resulted in a significant demand for cDNA clones, antibodies, expression vectors, etc., all in short order. Providing reagents for the expansion of research on such genes is scientifically important and potentially rewarding if the market catches up. Allele Biotech works diligently to supply the most up-to-date and active researchers with pre-validated viral particles for expressing many of these “hot” genes, (e.g. iPS factors, light-activated ion channels, factors that induce neuronal cells from other cell types). Allele Biotech produces custom-packaged high titer lentivirus and retrovirus using unique technologies, as described under “Services by Category”-> “Viral Packaging”.
Under the Essential Virus Grant Program, Allele Biotech accepts custom viral packaging orders, including cDNA of shRNAs against recently established critical mammalian factors. The process begins with an evaluation of your one page application explaining why the factor can potentially be in demand by other researchers. If the application is accepted, Allele Biotech will waive the charges for the project and consider it an R&D effort. Allele Biotech will also offer it as a shelf product at a much lower cost than custom projects. Interested researchers please submit your applications with your order; our accounting department will promptly forward your request to our review group.

New Product of the Week 05-03-10 to 05-09-10: Essential Virus Grant Program

Promotion of the Week 05-03-10 to 05-09-10: for a limited time, pre-packaged, validated IL2 and IL15 lentiviruses. http://www.allelebiotech.com/shopcart/index.php?c=206&sc=0

Tags: cDNA, essential genes, lentivirus, miRNA, promoter reporter, virus packaging