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

LoxP 4-in-1 iPS Factor on Lentiviral Vectors for Efficient Reprogramming

Putting 4 iPS factors on one lentiviral vector, separated by 2A peptides, has appeared to be more efficient in generating iPS cells than having all 4 factors on individual viruses, at least in a number of cases. Stem cell-like colonies start to appear in about 2 weeks using Allele Biotech’s 4-in-1 lentivirus. In addition to the concerted effects from Oct3/4, Sox2, c-Myc, Klf4, it is also believed that the coordinated silencing of these factors after reprogramming help forming iPS colonies.

The 4-in-1 lentivirus from Allele Biotech contains loxP sites that can be used to remove the 4 cDNAs if so desired. For convenience, a new product kit is offered starting this week to include lenti-nCre in a kit with the 4-in-1 iPS viral products.

New Product of the Week 04-11-10 to 04-18-10: 4-In-One-Vector: Human OSKM Lentiviral Paticles (Oct3/4, Sox2, Klf4 and c-Myc) and Cre Lentiviral Particle kits, Cat # ABP-SC-LVI4IN1C1 or ABP-SC-LVI4IN1C5

Promotion of the Week 04-11-10 to 04-18-10: Single vial 4-in-1 is offered only this week. This product has been well established and validated, one of the reasons smaller packages are not normally offered. As a matter of fact, every batch of the 4-in-1 iPS lentivirus has been sold out.

Update note: Lentivirus inserts into the host chromosome, and is gradually being replaced by footprint-free reprogramming reagents, the best being Allele Biotech’s enhanced mRNA reprogramming factors that feature a patent-pending fusion gene.

Tags: 4-in-1, c-myc, cre, iPS, iPSCs, Klf4, lentivirus, loxP, Oct3, repgramming, silencing, Sox2

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

Choosing siRNA, shRNA, and miRNA for Gene Silencing

RNAi refers to dsRNA-induced gene silencing, a cellular process that degrades RNA homologous to one strand of the dsRNA [1, 2]. The intermediates of long dsRNA-initiated RNAi are double-stranded small interfering RNAs (siRNA), typically 21-23 nucleotide (nt) long. The siRNAs, when introduced into cells, can be used to silence genes in mammalian systems where long dsRNAs prompt protein kinase R (PKR), RNase L, and interferon activities that result in non-specific RNA degradation and general shutdown of protein synthesis [3]. siRNAs can either be chemically synthesized then directly transfected into cells or can be generated inside the cell by introducing vectors that express short-hairpin RNA (shRNA) precursors of siRNAs. The process of shRNA into functional siRNA involves cellular RNAi machinery that naturally process genome encoded microRNAs (miRNA) that are responsible for cellular regulation of gene expression by modulating mRNA stability, translation, and chromatin structures [4].

Chemically synthesized siRNA is the simplest format for RNAi. One of the biggest hurdles for achieving effective RNAi with siRNA is that many cells are difficult to transfect. An RNAi experiment is typically considered successful when the target gene expression is reduced by >70%, a threshold not reachable by many types of cells due to their low transfection efficiency. Another drawback of using synthetic siRNA is the limited duration of post-transfection effects, typically with gene silencing activities peaking around 24 hours, and diminishing within 48 hours [5]. Chemical synthesis of siRNA, which is a service Allele Biotech and Orbigen (now merged under the Allele brand) pioneered and still provides, is expensive on a per transfection basis relative to DNA vector based reagents.

shRNA can be introduced by DNA plasmid, linear template, or packaged retroviral/lentiviral vectors. Using any form of DNA construct, except the PCR template format such as Allele’s LineSilence platform, requires creating DNA constructs and sequence verification; a taxing work load if multiple genes need to be studied. However, once the constructs are made, they can be reproduced easily and inexpensively. It is difficult to directly compare the effectiveness of siRNA versus shRNA on a per molecule basis because RNA polymerase III (Pol III) promoters such as U6 or H1 commonly used to express shRNAs can make thousands of copies of shRNA from a single DNA template. However when both siRNA and shRNA are produced the same way, e.g. synthesized chemically, shRNA is reported to be somewhat more effective [6, 7]. For the goals of this research, the most important advantage using shRNA can provide over siRNA is that it can be carried on a lentiviral vector and introduced into a wide variety of cells.

Similar to the comparison between siRNA versus shRNA, it is also difficult to rank the efficiency of shRNA versus miRNA from published data, partly due to different results from different experimental systems. There have been several reports that showed shRNA can cause significant cell toxicity, especially in vivo such as after injection into mouse brain. It was originally reasoned that highly efficient expression from Pol III promoters might overwhelm the cellular machinery that is needed to execute endogenous RNAi functions such as transporting miRNA from the nucleus to the cytoplasm. It was later found out that even using Pol III promoter to create miRNA could still mitigate the toxic effects of shRNA [8]. Since shRNA and miRNA are processed by endonuclease Dicer before being incorporated into RNA induced silencing complex (RISC), the exact identity of siRNAs produced from a given shRNA or miRNA targeting the same region on the mRNA are not known in most of the earlier studies. By designing shRNA and miRNA to give exactly the same processed siRNAs, Boudreau et al. showed that shRNA is actually more potent than miRNA in various systems [9].

New Product/Service of the Week (02-01-10 to 02-07-10): Lentrivirus retrovirus shRNA Packaging Services as low as under $900 per virus.

Currently Trendy Product Line: Camelid antibody group against fluorescent proteins as precipitation tag for co-IP (replacing formerly GFP-Trap line)–GFP-nAb, promotion ongoing now.

1. Fire, A., S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver, and C.C. Mello, Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 1998. 391(6669): p. 806-11.
2. Hannon, G.J., RNA interference. Nature, 2002. 418(6894): p. 244-51.
3. McManus, M.T. and P.A. Sharp, Gene silencing in mammals by small interfering RNAs. Nat Rev Genet, 2002. 3(10): p. 737-47.
4. Hutvagner, G. and P.D. Zamore, A microRNA in a multiple-turnover RNAi enzyme complex. Science, 2002. 297(5589): p. 2056-60.
5. Rao, D.D., J.S. Vorhies, N. Senzer, and J. Nemunaitis, siRNA vs. shRNA: similarities and differences. Adv Drug Deliv Rev, 2009. 61(9): p. 746-59.
6. Vlassov, A.V., B. Korba, K. Farrar, S. Mukerjee, A.A. Seyhan, H. Ilves, R.L. Kaspar, D. Leake, S.A. Kazakov, and B.H. Johnston, shRNAs targeting hepatitis C: effects of sequence and structural features, and comparision with siRNA. Oligonucleotides, 2007. 17(2): p. 223-36.
7. Siolas, D., C. Lerner, J. Burchard, W. Ge, P.S. Linsley, P.J. Paddison, G.J. Hannon, and M.A. Cleary, Synthetic shRNAs as potent RNAi triggers. Nat Biotechnol, 2005. 23(2): p. 227-31.
8. McBride, J.L., R.L. Boudreau, S.Q. Harper, P.D. Staber, A.M. Monteys, I. Martins, B.L. Gilmore, H. Burstein, R.W. Peluso, B. Polisky, B.J. Carter, and B.L. Davidson, Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: implications for the therapeutic development of RNAi. Proc Natl Acad Sci U S A, 2008. 105(15): p. 5868-73.
9. Boudreau, R.L., A.M. Monteys, and B.L. Davidson, Minimizing variables among hairpin-based RNAi vectors reveals the potency of shRNAs. Rna, 2008. 14(9): p. 1834-44.

Tags: alpaca antibodies, camelid antibody, gene knockdown, gene silencing, GFP-Trap, lentivirus packaging, miRNA, nanobodies, packaging systems, RFP-Trap, RNAi, shRNA, shRNA packaging, siRNA, VHH, virus packaging service

Commonly Known Facts About Viral Packaging -That Might Not Be Correct…

Packaging lentiviruses or retroviruses is not a routine procedure that every biology lab performs even if there is need to use it. A viral packaging protocol normally begins with preparation of purified transfer plasmid DNA, a miniprep should be enough for a few transfections. The virus backbone plasmid is either co-transfected into commonly used cells with helper plasmids that provide the essential proteins required for particle packaging, or transfected into established helper cell lines that express the required proteins from integrated transgenes. After incubation of packaging cells for a couple of days, viruses are collected and tittered. Titer determination is somewhat tricky for the inexperienced. Using a control virus expressing a fluorescent protein can make this step convenient.

Commonly known facts:

1) Lentiviruses are packaged at a titer of 10^6 IU/ml without concentrating steps.

This needs update since with more advanced technologies lentiviruses can be packaged routinely at 10^8 IU/ml. With further concentrating, the titer can be easily above 10^11 IU/ml. Retroviruses can be packaged to similar titers as well.

2) Using packaging cell lines gives the highest possible titer

While packaging cell lines (such as Allele’s popular Phoenix Eco and Ampho cells for retrovirus packaging) provides maybe the most convenient method for packaging, the yield will not reach the highest potential. Packaging cell lines may also lose their capability for packaging after continued culturing, requiring periodic selection with antibiotics and functional tests, as we do here at Allele.

3) Retroviruses are always collected in one shot after transfection into packaging cells

If the transfer vector has oriP/EBNA1 episomal maintenance system, such as some of the Phoenix vectors Allele offers, the plasmids may continue to express for up to 30 days. With puromycin selection, the titer of retrovirus produced from Eco or Ampho cells can reach 10^7 IU/ml.

This week’s promotion (102509-103109): 10% off across the board of Allele Biotech’s custom services, for an example, check out our world-leading baculovirus protein expression.

New Product/Service of the Week: Introduction of Custom Viral Packaging Service. Routine titer of 10^8 IU/ml, as high as 10^10 IU/ml, option to include cloning. Signature service ABP-CS-MERV002 provides more than 200 million particles at $7/million particles. These are game-changing prices for the viral packaging service market based on superior technologies!

Tags: baculovirus, co-transfection, custom service, iPS lentivirus, iPS retrovirus, lentivirus, packaging cells, protein expression, retrovirus, viral packaging, viral particles, viral vectors, virus titers