New Frontiers for Research Tool Development in the New Year

Allele Biotech's Green Crystal Ball

Optogenetics
Chosen as the Method of the Year 2010 by Nature Method and mentioned in a number of year-end recaps, this is a technology that allows the use of light to precisely (at least in a temporal sense) control engineered proteins within a targeted cell population. For example, by introducing light-activated channelrhodopsins into neurons, one can use a pulse of light to initiate a movement of ion across the cell membrane. The technology, first reported in 2005 then made headlines as a major impact on neurosciences since 2007, is now being combined with other components in controlling a broader array of biological events, such as DNA binding, enzyme activities, etc. Looking forward, a few areas will be more than likely the frontlines of moving optogenetics into more labs:

Additional combinations: The few known channelrhodopsins and their fast growing variations will be combined with more “effecter” domains to control different events. The challenge will be to find ways to use the structural changes or any responses channelrhodopsins have to stimulating lights in order to trigger a reaction in the associated effecter domain.

Tracking mechanisms: A platter of fluorescent proteins (FPs) will be used as an independent tracking method to follow cells being targeted. FPs that have optical spectra that do not interfere with the optogenetic molecules will be tested and established. In addition, FPs with less toxicity, narrower excitation and emission peaks, and more tolerance to different cellular environment will be preferred and eventually set up as standards.

Delivery tools: To bring the optogenetic reagents into cells like neurons researchers will most likely rely on lentiviral vectors in most cases. Other vehicles such as baculovirus, MMLV-based retrovirus, even herpes virus may find broader applications in this field. Pre-packaged lentiviruses and MMLV-retroviruses already contain optogenetic constructs will become popular products.

VHH Antibodies
The small capture polypeptides based on single-domain Camelid antibodies (nanobodies, nano antbodies or nAbs) and similar VHH domains will become much dramatically more popular this year, judging from the significant increase in demands of the only camelid reagent products, GFP-Trap and RFP-Trap, in 2010. There are a number of NIH initiated programs that aim to find capture reagents that eventually target the complete human proteome. One of the key criteria for the current phase of the relevant NIH Director’s Initiative is ability to co-immunoprecipitate. The Human Proteome Organization (HUPO) recently expressed frustration due to the lack of high quality capture reagents necessary to isolate and identify most proteins. HUPO promotes global research on proteins in order to decode the human proteome. From what we have learned from dozens of publications showing the use of GFP-Trap, VHH molecules pulls down GFP-tagged proteins with unprecedented efficiency and purity. VHH antibodies show strong affinity and specificity, at a level superior or comparable to monoclonal antibodies. In addition, VHH antibodies are increasingly appreciated for their capabilities to recognize concave epitopes by their relatively convex-shaped paratopes. VHH nanobodies are small (~12-15 kD), with a limited number of functionally important disulfide bonds, can be expressed very well in E. coli, and are amazingly stable in extreme denaturing conditions such as heat and acid. They have been shown to be better suited for in vivo and trans-cellular membrane delivery than other antibodies. It should not be surprising that one day in the coming years VHH antibodies will be more dominant than monoclonal antibodies.

Super-Resolution Imaging
One of the goals of developing technologies such as photoactivated localization microscopy (PALM) and related super-resolution imaging (SRI) techniques was to achieve electron microscopy (EM) level resolution without using EM. Now new developments show that maybe combining EM and photoactivable FPs would provide more specific and more detailed morphology. It would be anticipated that more photoconvertible FPs will prove to work well for one type of SRI or another. The event that will bring this technology to nearly every cell biology lab is the improvement and availability of necessary instruments that some companies have already begun to commercialize.

New Product of the Week 010311-010911:

Human let-7b miRNA minigene on lentivirus with RFP reporter, ABP-RP-MILT7BLP

Promotion of the Week 010311-010911:

15% off mWasabi-based organelle markers carried on baculo2mammalian system if order this week (On-Demand products will require about 3-4 weeks for virus packaging after an order is placed). Use code 0103BACFP on fax or email order.

Tags: alpaca antibodies, camelid antibodies, channelrhodopsin, EM, halorhodopsin, optogenetic, PALM, photoactivable FP, photoconvertible FP, STED, STORM, VHH, VHH antibodies

Working on the Wnt signaling pathway

Wnt proteins form a family of highly conserved, secreted glycolipoproteins that regulate cell proliferation, cell polarity and cell fate determination during embryonic development and tissue homeostasis. Mutations in Wnt genes or the Wnt signaling pathway components are often linked to human birth defects, cancer and other diseases.

Since the discovery of the Wnt-1 gene 27 years ago, a complex Wnt signaling network with many components having multiple distinct roles and acting in different cellular compartments has been established. The studies of Wnt signaling in human diseases, in stem cell biology and tissue regeneration holds promise for translational medicine.

A few commonly asked questions regarding studies on the Wnt pathways will be touched upon in this blog series:

How to activate the Wnt Signaling Pathway?

1) Recombinant Wnt Proteins: there are several commercial sources for recombinant Wnt proteins. As most of them are produced in bacteria, endotoxin, purity and active concentration are of primary concern. From our own experience using commonly used commercial Wnt proteins, the activities vary from batch-to-batch, and sometimes the products are simply inadequate.

2) Conditioned Medium: this approach is convenient and of low cost. ATCC provides two mouse fibroblast cell lines, which over-express mouse Wnt3A (CRL-2647) and mouse Wnt5A (CRL-2814). Researchers can maintain the cell lines and collect conditioned medium whenever they need Wnt proteins. However, these are the only two Wnt over-expressing cell lines available on the market, yet there are 19 members in the Wnt gene family. If you are planning on working with Wnt proteins other than 3A and 5A, you would have to develop relevant cell lines on your own. On the other hand, because over-expression of Wnt proteins will also activate the secretion of some growth factors, such as FGFs, the conditioned medium should be a mixture of several kinds of secreted factors.

3) Over-expression Wnt proteins directly in your cells of interests: Over-expression is a basic strategy for functional biology research. One of the barriers for introducing cDNAs into cells is the transfection efficiency. The advent of retroviral transfection (MMLV based or Lenti based) technically resolves this problem, as the transduction efficiency can reach almost 100%. However, highly efficient retrovirus packaging remains a difficult process for most research labs.

Allele Biotech plans to introduce all the human Wnt cDNAs on its HiTiter™ Expression Lentivirus platform as shelf products. The package size will be 5 vials, each vial 100 µl, the titer is 10e8 TU/ml at a price of $799. This product line will be under “Product-on-Demand”, with clones ready but packaged only upon order for the first time. First-time orders will qualify for a 10% discount, if the first-time customer provides the cDNA, there will be an additional 20% discount. These lentiviruses can be used directly in target cells or to build over-expressing cell lines for conditioned medium. Next topic is How to inhibit Wnt signaling pathway?

New Product of the Week 120610-121310: Human miRNA minigene on lentivirus with RFP reporter, ABP-RP-MI221LP

Promotion of the Wee 120610-121310: 5% off virus packaging if you use our FaceBook code “ViralPackaging” at shop.allelebiotech.com (beta-test new web).

Tags: cell fate, cell polarity, cell proliferation, conditioned medium, growth factors, lentivirus, signaling pathways, Wnt

Happy Holidays!

We like to explore and are inspired by journeys to the unknown. Science is not only a disciple of reason, but also one of romance and passion.

Physicist Stephen Hawking, in a Parade magazine interview, September 12, 2010

New Product of the Week 122010-122610:

pORB-CAG-AFP-GFPfusion(mWasabi)-sIRES-VSVG for insect and mammalian cell expression of human cancer antigen protein AFP, validated and ready to ship. Email vivec@allelebiotech.com

Promotion of the Wee 122010-122610:

5% off all cell culture related products including consumables. For this week use the code CellCulture when you check out at our website (http://shop.allelebiotech.com/).

Tags: science

How to order virus packaging service

Step 1: Please review the following before ordering Allele’s retrovirus packaging service.

Verify that your plasmids for virus production meet the following criteria:

No internal polyA signal or polyA signal immediately downstream of the coding cistron.

Avoid toxic genes, unusual secondary structures, and expression cassettes > 8kb (5’ to 3’ LTR).

If these concerns can not be satisfied in your vector design, please contact our technical specialist (vivec@allelebiotech.com) for further discussion, as Allele’s proprietary packaging technology might be able to help.

Step 2: Select the service:

Allele offers multiple project discounts: a 5% discount for ordering 3-5 packaging services, and a 10% for 6-10 packaging services.

Additional information can be found here.

Step 3: Send us 10 ug of endotoxin-free plasmid (blue ice shipping) for virus packaging.

To facilitate the completion of the custom virus packaging projects, Allele offers a variety of retro/lentivirus plasmids as well as viral plasmid subcloning and endotoxin-free plasmid preparation services. Please contact us (oligo@allelebiotech.com or 858-587-6645) for further details.

Once your order is placed, we will contact you about plasmid shipment and provide you with a time frame for completing the project.

Note: If the total project price is more than $1000, a non-refundable down payment (30% of the total service price) is registered before the project is initiated.

New Product of the Week 121310-121910:

pORB-ICAM/mWasabi-sIRES-VSVG for insect and mammalian cell expression of human membrane protein ICAM, validated and ready to ship. Email vivec@allelebiotech.com

Promotion of the Wee 120610-121310:

5% discount on high quality cell culture dishes manufactured by Phoenix Biomedical, email oligo@allelebiotech.com with code PH121310EC.

Tags: custom virus packaging, EF1a, lentivirus, lentivirus packaging, LTR, retrovirus, retrovirus titering, SV40

How to titer retrovirus/lentivirus

A number of methods have been developed for determining the lentiviral/retroviral titers such as using ELISA to assess the amount of p24 antigen, enzyme assay to quantify reverse transcriptase activity, and real time RT-PCR to count viral RNA genome. However, these methods do not differentiate non-infectious from infectious virus particles that are present in the virus preparation; therefore, these measurements rely on some correlation factors to derive a titer, which can be inaccurate.

Because an accurate virus titer is critical for most experimental purposes, Allele Biotech’ pre-packaged viruses are measured by the number of genomic integration events to determine the infectious titer. This functional titering is done by first transducing TE671cells (selected for consistent transduction efficiency) followed by measuring the viral genome integrated into the cell genome with QPCR. The number of cellular genome copies is also measured, and the copy number of viruses is divided by that of cells to obtain active virus titer.

A plot of quantitative PCR (Q-PCR) titering of virus preparation is provided above.
TE671 cells (1×105) were transduced with 2×10-3ml of a virus sample to be titered. Cells were harvested and DNA extracted for QPCR. The titer is determined as followed, transduction unit per ml (TU/ml)=N [X1/(X2/2)]/2×10-3ml, where N is the cell number, X1 is the copy number of integrated virus DNA, and X2 is the copy number of GAPDH DNA.

New Product of the Week 120610-121310:

CR4(Oct3/4)-ResponseElement-mClavGR2 Reporter Pre-packaged Lentivirus. Shows Oct3/4 target gene transcription by following a photoconvertible FP in real time. Product-on-demand. Email iPS@allelebiotech.com for details.

Promotion of the Wee 120610-121310:

Buy one FRET oligo at $200 (50nmol HPLC purified), get the second at half price. Email order to oligo@allelebiotech.com with code AB120710SO.

Tags: Allele Biotech lentivirus, infectious virus titering, lentivirus titering, p24 ELISA, retrovirus titering, viral genome RT-PCR, virus packaging, virus titering

Wednesday, December 8th, 2010 Viruses and cells No Comments