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

Features of Allele’s Virus Packaging Services

High Titer
Allele Biotech’s HiTiter™ Viral Packaging Services, using proprietary technologies that are unique and highly efficient, can easily yield 10e8 to 10e9 TU/ml lentivirus/retrovirus without concentrating steps. With a concentration step 10e9 to 10e10 TU/ml can be achieved, which is ideal for in vivo research. It’s the world’s most powerful viral packaging platform.

Quick Turnaround
Allele Biotech has consolidated its procedure of HiTiter™ Viral Packaging Services as a standard production-line resulting in a quick turnaround. Typically, a standard 10e8 TU/ml 2ml packaging service is completed in one week and delivered in less than two weeks. You send us virus transfer plasmids, wait a week or two, and receive high titer ready-to-use virus. Imagine how our ingenuity and innovation can pave the way for your research!

Ideal for Complicated Constructs
Viral packaging, especially for complicated constructs, has been intrinsically difficult for most researchers. Now with Allele Biotech HiTiter™ Viral Packaging Services, you have an ideal option for packaging even the most demanding constructs! We have successfully developed the 4-In-1 iPSC Generation Lentivirus, expressing 4 genes (hOct3/4, hSox2, hKlf4 and hc-Myc) in one lentiviral construct, which has been very popular in the filed of stem cell research.

Fluorescent Proteins & Drug Resistance
Allele Biotech and our collaborators design, evolve, and select new FPs that suit different applications. These brighter fluorescent proteins, sometimes together with drug resistance genes, have been incorporated into our lentiviral/retroviral systems. When you order our viral packaging service, you will automatically receive a 20% discount for all our listed viral plasmids.

Full RNA Interference Services
We have RNAi lentiviral packaging services, RNAi validation services, and RNAi screening services, which can meet different demands in the filed of RNA Interference. The RNAi lentiviral packaging services include Gene-To-Silence™, Sequence-to-Virus™ and DNA-to-Virus™. The plasmids we use for these services have build-in FPs and drug resistance genes, convenient for virtually all research purposes.

New Product of the Week 111510-112110:

Cre/loxP Reporter Cell Line LoxP-lacZ Human Fibroblast, ABP-RP-CLACLOX.

Promotion of the Wee 111510-112110:

Fine agarose for DNA or RNA gel, buy 500g, get a second bottle of agarose at 40% off, use promo code FB111510AR, order by emailing oligo@allelebiotech.com

Tags: custom viral packaging, high titer, lenti-cre, lentivirus, packaging lentivirus, pre-packaged viruses, retrovirus, RNAi screening

Expression of iPS Factors from Transfected mRNA

Differentiated cells can be reprogrammed to pluripotency by enforced expression of certain combinations of stem cell-specific protein factors in them. The power of this method was first demonstrated by Yamanaka’s group using retroviruses carrying Oct3/4, Sox2, c-Myc, and Klf4. Alternative factors such as Lin28 and Nanog, and additional factors such as the human telomerase gene hTert and shRNA against p53 were also shown to contribute to reprogramming. From the very beginning it was realized that viral integration would pose a major problem in using the induced pluripotent stem cells (iPSCs) for clinical purposes. There have been multiple attempts to circumvent this problem by using non-integrating vectors such as plasmid, minicircle DNA, adenovirus, baculovirus, removable transposons, episomal DNA, or by introducing recombinant proteins with a transmembrane domain into target cells. From reports in the field and customer feedbacks it seems that retroviral or lentiviral systems are still the most efficient in reprogramming. mRNA is about the only option left unreported, until an article by Warren et al was published in Cell Stem Cell online recently.

From that report, it is clear that the reason that it took so long for RNA-induced iPSCs (RiPSCs) to appear in the literature was because synthetic mRNAs activate interferon responses in mammalian cells, reminding us of the early days of RNAi. The authors took a number of steps to reduce interferon responses, including adding a 5’-cap (actually a fairly standard step in in vitro transcription), using a phosphatase to remove 5’ triphosphates on uncapped mRNAs, and using modified C and U bases (5-methucytidine or 5mC and pseudouridine or psi) during T7 promoter-driven in vitro transcription. The prepared mRNA was then administered everyday for 17 days at an amount not clearly defined in the paper. The main benefit of this method is of course that there is no gene integration to alter the chromosome. The efficiency of the new method was also compared to using viral vectors and it was shown that 1.4% conversion efficiency was achieved vs retroviral systems’ 0.01% (although we have experienced better results using lentivirus, at least the 4-in-1 version).

The DNA templates used for in vitro transcription of the iPS factors were created by multiple PCR reactions and bridged ligation; it could also be done by other cloning strategies. For those excited about trying this new way of making iPSCs, the major hassle would be preparing modified mRNAs good and abundant enough for 17 consecutive transfections. Allele Biotech would like to provide custom services, before offering shelf products, for creating such mRNAs as the method sounds potentially very helpful to many researchers in the iPSC field.

New Product of the Week 100410-101010:

pLICO-mWasabi (Promoterless FP Reporter Vector ), listed as product-on-demand, now available, ABP-HL-PE40010 $395.00.

Promotion of the Week 100410-101010:

Barrier too high to start using virus? Allele lowers it for starters, $500 for bactulo virus protein production, and $300 retrovirus packaging. Code 100310VIVEC, email vivec@allelebiotech.com

Tags: c-myc, in vitro transcription, integration-free, iPSC, iPSCs, Klf, lentivirus, modified synthetic mRNA, mRNA iPSCs, mRNA transfection, Oct, retrovirus, RiPSC, RiPSCs, Sox

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

Retroviral Vectors with Integrated oriP/EBNA1 for IPSC

The new product of the week of Sep 21-27 is the retrovirus plasmid sets that contain a built-in episomal expression system. As we have discussed previously, OriP/EBNA1 system originated from Epstein-Bar virus, which allows the establishment of stable episomes at 5-20 copies per cell, and duplication once per cell division.

By using the oriP/EBNA1 episomal system, reprogramming cDNAs can be expressed at prolonged time period in reference to plasmid transfection, without integration into chromosomal DNA. A paper published in PLoS One on Sep 18, 2009 by Marchetto et al. showed that by using such a system (on different plasmids) the authors were able to create induced pluripotent stems cells (iPS cells,) effectively from human embryo neural precursor cells.

The Allele pCHAC-EBNA system has dual functions: it can be ready-to-use plasmids for episomal expression of Oct4, Sox2, c-Myc, Klf4, or Nanog and Lin28 by a simple transfection into target cells; it can also be packaged into retroviruses by transfecting into the Allele Phoenix Retrovirus packaging Eco or Ampho cells. This product group is officially launched today. It should become a highly convenient and unique tool for iPSC-related studies.

Tags: Ampho cells, EBNA, Eco cells, episomal expression, iPS cells, iPSC, iPSCs, New Product of the week, oriP, Phoenix Retroviral System, reprogramming, retroviral vector, retrovirus