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

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

Delivery of RNAi or Cre by Ultrasound-Guided Injection of High Titer Lentiviral Vectors

By Jiwu Wang

According to the Skin Cancer Foundation, skin cancer is the most common type of cancer in the US. Although the skin might seem to be an easy target for gene therapy or RNAi mediated functional corrections, the outer keratinized epithelial cells forms a formidable barrier to delivery of genetic material. The epidermis undergoes rapid turnover, a fact that further complicates gene therapy because gene transfer to skin stem cells would be required for sustained effects.

Before skin gene therapy can be discussed with any practical meaning, a physiologically relevant in vivo model for studying gene function in the context of tumorigenesis and epithelial biology must be established. Studies of gene functions in skin homeostasis in mouse models were mostly performed by labor-intensive knockout methods. Recently, at least two publications have shown that by using ultrasound-guided injection of lentiviruses into amniotic fluids, transgene or shRNA can be efficiently and specifically delivered to epidermis, including skin stem cells, creating a very attractive model for functional studies and therapeutic tests.

Localized injection of high titer lentiviral vectors has been widely used for studying genes in brain development and a few other areas. Instead of injection into animal tissues, Endo et al. injected tiny volume (nl) of high titer lentivirus (10e10 TU/ml) into amniotic cavities within a defined window of embryogenesis [1]. By following fluorescent protein markers (CFP, GFP, YFP, RFP), both Endo et al. and researchers from Elaine Fuchs group demonstrated high efficiency and specificity of delivery to epithelial cells, commonly resulting in multiple genomic insertions of the viral genome.

RNAi against alfa1-catenin was used by Beronja and colleagues as an example to show that loss-of-function analysis can be done rather easily using shRNA/FP bearing lentivirus [2]. nlCre was also delivered to embryos with loxP-flanked transgenes vs wildtype for conditional knockout studies. These new findings should open doors to various experiments and therapies concerning the health of the skin.

1. Endo, M., P.W. Zoltick, W.H. Peranteau, A. Radu, N. Muvarak, M. Ito, Z. Yang, G. Cotsarelis, and A.W. Flake, Efficient in vivo targeting of epidermal stem cells by early gestational intraamniotic injection of lentiviral vector driven by the keratin 5 promoter. Mol Ther, 2008. 16(1): p. 131-7.
2. Beronja, S., G. Livshits, S. Williams, and E. Fuchs, Rapid functional dissection of genetic networks via tissue-specific transduction and RNAi in mouse embryos. Nat Med. 16(7): p. 821-7.

New Product of the Week

080210-080810: mTFP1-Mitochondria-neoR plasmid, a new drug-resistant version of Allele’s organelle markers

Promotion of the Week

080210-080810: Retroviruses expressing OSKM or OSNL set of iPS factors at $500 for order placed this week only for all Allele Facebook fans, others with code iPS0808 mentioned in order.

Tags: cre, Fluorescent proteins, in vivo injection, lentiviral vector, lentivirus, loxP, nlCre, RNAi, shRNA, skin cancer, skin care, skin therapy, sunscreen, transgene

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