iPSCs Generation Viral Particles

From the worlds most powerful viral packaging platform. Introducing cost effectiveness to your research is just the beginning. Imaging how our ingenuity and innovation can pave the way for your research.
he direct reprogramming of somatic cells to pluripotency was rst accomplished in 2006. Adult mouse broblasts were converted to iPS Cells (induced pluripotent stem cells) through ectopic expression of a select group of transcription factors. In 2007, direct reprogramming was achieved in human cells. The generation of iPS from differentiated adult cells has vast therapeutic implications, particularly in the context of pharmaceutical screening and cellular replacement therapies. Extensive research has been conducted in search of efcient ways for iPSCs generation. Lentiviral vectors were used in the original iPSC experiments and remain to be the most popular method. Its advantages over other gene-delivery methods include high-efciency infection of dividing and non-dividing cells, long- term stable expression of transgenes, and low immunogenicity. Allele Biotech provides custom retrovirus or lentivirus packaging services using proprietary technologies that are unique and highly efcient, yielding 10^8 to 10^9 TU/ml, without any concentrating steps. These technologies and optimal operation procedures enable Allele Biotech to package viruses at < 1% of the market price in certain categories on per million particle basis. Based on the most powerful viral packaging platform, Allele Biotech offers Pre-packaged iPSCs Generation Lentiviral Particles, which are validated, ready-to-use, high quality

For more information visit www.allelebiotech.com

reagents for any laboratory to create iPS cells. A straight-forward and optimized protocol is also provided for immediate use of these products. In this system, 4 product lines are available: 1) Basic Lentiviral Particles: lentiviruses carrying each of the 6 reported reprogramming factorshOct3/4, hSox2, hKlf4, hc-Myc, hNanog and hLin28. Or enhancing factors p53 shRNA and hTert are provided individually or in combination sets. 2) Lentiviral Particles Co-expressed with Reporter: uorescent proteins are co-expressed from the iPS factor carrying lentivirus to trace their expression during the reprogramming procedure. 3) Lentiviral Particles Polycistronic Expression with loxP Sites. Polycistronic expression of hOct3/4, hSox2, hKlf4 and hc-Myc, in one lentiviral vector, referred as 4-In-1, was reported to reprogram efciently. More recently, polycistronic expression of only two of the essential factors, hOct3/4 and hSox2, referred as 2-In1, is gaining popularity in iPSC generation. Meanwhile, it seems benecial to silence the exogenous iPS factors after the initiation of reprogramming. Allele Biotech combines all these advancement in iPSCs eld and provides both 4-In-1 and 2-In-1 with loxP sites by which the iPS factors may be removed with a Cre recombinase. 4) Basic Retroviral Particles: MMLV-based retroviruses carrying

each of the 6 reported reprogramming factors, hOct3/4, hSox2, hKlf4, hc-Myc, hNanog and hLin28 are provided individually or in combination sets. Retroviral systems are preferred by some iPSC researchers because retrovirus is silenced by the host more efciently than lentivirus after the trangenes exert their effects. References: 1. Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult Fibroblast cultures by dened factors. Cell 126, 663676. 2. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. (2007). Induction of pluripotent stem cells from adult human broblasts by de ned factors. Cell 131, 861872. 3. Maherali, N., and Hochedlinger, K. (2008). Guidelines and techniques for the generation of Induced pluripotent stem cells. Cell Stem Cell 3,

or Research Use Only. Not for Diagnostic or Therapeutic Use. Purchase does not include or carry any right to resell or transfer this product either as a stand-alone product or as a component of another product. Any use of this product other than the permitted use without the express written authorization of Allele Biotech is strictly prohibited

Patent Issuses Lentiviral vector design and virus packaging techniques used to create this product line are covered by several United States patents under agreement to Allele Biotech, contact vivec@allelebiotech.com or call 858-587-6645 for details. Safety Issuses Lentiviral vectors should be handled using NIH BSL-2 safety guidelines. For more information, please see Biosafety in Microbiological Laboratories <4th edition> which is available on the National Institutes of Health website at http://bmbl.od.nih.gov

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Basic Lentiviral Particles

Lentiviruses carrying each of the 6 reported reprogramming factors, hOct3/4, hSox2, hKlf4, hc-Myc, hNanog and hLin28, as well as enhancing factors p53 shRNA and hTert, are provided individually or in combination sets. These are ideal for researchers who would like to transduce reprogramming factors into cells without introducing any uorescence or selection markers. The effects of individual factors or various ratios among the factors may be tested by using this group of products. Results have demonstrated that inhibiting cell cycle regulator p53 with shRNA or over-expressing telomerase gene hTert may increase the efciency of iPSC generation, shortening the time otherwise needed to reprogram. Lentivirus expressing a single reprogramming factor is packaged in 5 vials per order. Each vial contains 10^7TU viral particles at 10^8TU/ml titer in a volume of 100ul. Lentivirus sets include 1 vial of each virus, with O, S, K, M, N, L, P, and T indicating hOct3/4, hSox2, hKlf4, hc-Myc, hNanog, hLin28, p53 shRNA and hTert, respectively.

Box 1 | Product List and Contents

Individuals hOct3/4 hSox2 hKlf4 hc-Myc hNanog hLin28 p53 shRNA hTert Sets Human OSKM Set Human OSKMP Set Human OSKMPT Set Human OSNL Set Human OSNLP Set Human OSNLPT Set ABP-SC-LVIOSKM ABP-SC-LVIOSKMP ABP-SC-LVIOSKMPT ABP-SC-LVIOSNL ABP-SC-LVIOSNLP ABP-SC-LVIOSNLPT each gene 10^7TU in 1 vial 0.1ml, 4 vials each gene 10^7TU in 1 vial 0.1ml, 5 vials each gene 10^7TU in 1 vial 0.1ml, 6 vials each gene 10^7TU in 1 vial 0.1ml, 4 vials each gene 10^7TU in 1 vial 0.1ml, 5 vials each gene 10^7TU in 1 vial 0.1ml, 6 vials ABP-SC-LVIOCT3 ABP-SC-LVISOX2 ABP-SC-LVIKLF4 ABP-SC-LVICMYC ABP-SC-LVINANO ABP-SC-LVILIN28 ABP-SC-LVIP53S ABP-SC-LVITERT 5 vials, 10^7TU in 0.1ml per vial 5 vials, 10^7TU in 0.1ml per vial 5 vials, 10^7TU in 0.1ml per vial 5 vials, 10^7TU in 0.1ml per vial 5 vials, 10^7TU in 0.1ml per vial 5 vials, 10^7TU in 0.1ml per vial 5 vials, 10^7TU in 0.1ml per vial 5 vials, 10^7TU in 0.1ml per vial

Box 2 | Vector Maps

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Lentiviral Particles Co-expressed with Reporter

Fluorescent proteins are co-expressed from the lentiviruses carrying iPSCs factors to trace their expression during the re-programming procedure. Individual reprogramming factors will be paired with FPs of different spectra in this product group. Fluorescence generated from these lentiviruses can be used to trace infected cells. The expression of FP markers is regulated by viral genome silencing together with the transcription factors. Currently, hOct3/4-RFP is provided as OR. The other 3 constructs, depicted as grey in the vector maps, are under construction. Other factors may be coupled to FP markers by customer request. Lentiviruses expressing a single reprogramming factor are packaged in 5 vials per order. Each vial contains 10^7TU viral particles at 10^8TU/ml titer in a volume of 100ul. Lentivirus sets include 1 vial of each virus, OR, S, K, M , N, L, P and T indicate hOct3/4-RFP, hSox2, hKlf4, hc-Myc, hNanog, hLin28, p53 shRNA and hTert respectively. Once SF, KF and MF are ready, S, K and M will be updated.

Box 3 | Product List and Contents

Individuals hOct3/4-RFP (OR) hSox2-FP (S )
F

ABP-SC-LVIOCT3R Coming Soon Coming Soon Coming Soon

5 vials, 10^7TU in 0.1ml per vial 5 vials, 10^7TU in 0.1ml per vial 5 vials, 10^7TU in 0.1ml per vial 5 vials, 10^7TU in 0.1ml per vial

hKlf4-FP (KF) hc-Myc-FP (MF) Sets Human ORSKM Set (FP Plus) Human ORSKMP Set (FP Plus) Human O SKMPT Set (FP Plus)
R

ABP-SC-LVIOSKMF ABP-SC-LVIOSKMPF ABP-SC-LVIOSKMPTF ABP-SC-LVIOSNLF ABP-SC-LVIOSNLPF

each gene 10^7TU in 1 vial 0.1ml, 4 vials each gene 10^7TU in 1 vial 0.1ml, 5 vials each gene 10^7TU in 1 vial 0.1ml, 6 vials each gene 10^7TU in 1 vial 0.1ml, 4 vials each gene 10^7TU in 1 vial 0.1ml, 5 vials

Human ORSNL Set (FP Plus) Human O SNLP Set (FP Plus)
R

Box 4 | Vector Maps

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Lentiviral Particles Polycistronic Expression with loxP Sites

Multiple reprogramming factors are expressed from a single, polycistronic transcript into individual polypeptides through the function of 2A self-cleaving peptide sites. Either all four Yamenaka factors or two of the more critical factors, Oct3/4 and Sox2, are expressed from a single lentivirus vector. The cDNAs are anked by loxP sites that can be used to remove most of the viral genome by using a nuclear Cre construct, also provided in this product group. LacZ or RFP, which may be activated by nuclear Cre recombinase, are provided on control lentiviruses to test the functions of Cre in a particular type of target cell.

Box 5 | Product List and Contents

Polycistronic Expression with loxP sites Individuals 4-In-1: Human OSKM 2-In-1: Human OS 2-In-1-RFP: Human OS with RFP Sets 4-In-1 (2 vials) with p53 shRNA (2 vials) 4-In-1 (2 vials) with p53 shRNA (2 vials) and hTERT (2 vials) 4-In-1 (2 vials) with nlCre-GFP (2 vials) 4-In-1 (2 vials) with nlCre-GFP (2 vials) and loxP-RFP (1 vials) 4-In-1 (2 vials) with nlCre-GFP (2 vials) and loxP-lacZ (1 vials) 2-In-1-RFP (2 vials) with nlCre-GFP (2 vials) 2-In-1-RFP (2 vials) with nlCre-GFP (2 vials) and loxP-RFP (1 vials) 2-In-1-RFP (2 vials) with nlCre-GFP (2 vials) and loxP-lacZ (1 vials) ABP-SC-LVI4in1P ABP-SC-LVI4in1PT ABP-SC-LVI4in1C ABP-SC-LVI4in1CR ABP-SC-LVI4in1CZ ABP-SC-LVI2in1C ABP-SC-LVI2in1CR ABP-SC-LVI2in1CZ 4 vials, 10^7TU in 0.1ml per vial 6 vials, 10^7TU in 0.1ml per vial 4 vials, 10^7TU in 0.1ml per vial 5 vial, 10^7TU in 0.1ml per vial 5 vial, 10^7TU in 0.1ml per vial 4 vials, 10^7TU in 0.1ml per vial 5 vials, 10^7TU in 0.1ml per vial 5 vials, 10^7TU in 0.1ml per vial ABP-SC-LVI4in1 ABP-SC-LVI2in1 ABP-SC-LVI2in1R 5 vials, 10^7TU in 0.1ml per vial 5 vials, 10^7TU in 0.1ml per vial 5 vials, 10^7TU in 0.1ml per vial

Box 6 | Vector Maps

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Basic Retroviral Particles

MMLV-based retroviruses carrying each of the 6 reported reprogramming factors, hOct3/4, hSox2, hKlf4, hc-Myc, hNanog and hLin28 are provided individually or in combination sets. Retroviral systems are preferred by some iPSC researchers because retrovirus is silenced by the host more efciently than lentivirus after the trangenes exert their effecits. Lentivirus expressing a single reprogramming factor is packaged in 5 vials per order. Each vial contains 10^7TU viral particles at 10^8TU/ml titer in a volume of 100ul. Lentivirus sets include 1 vial of each virus, with O, S, K, M, N, L, P, and T indicating hOct3/4, hSox2, hKlf4, hc-Myc, hNanog, hLin28, p53 shRNA and hTert, respectively.

Box 7 | Product List

Individuals
hOct3/4 hSox2 hKlf4 hc-Myc hNanog hLin28 ABP-SC-LVIOCT3 ABP-SC-LVISOX2 ABP-SC-LVIKLF4 ABP-SC-LVICMYC ABP-SC-LVINANO ABP-SC-LVILN28 5 vials, 10^7TU in 0.1ml per vial 5 vials, 10^7TU in 0.1ml per vial 5 vials, 10^7TU in 0.1ml per vial 5 vials, 10^7TU in 0.1ml per vial 5 vials, 10^7TU in 0.1ml per vial 5 vials, 10^7TU in 0.1ml per vial

Sets
Human OSKM Set Human OSNL Set ABP-SC-LVIOSKM ABP-SC-LVIOSNL each gene 10^7TU in 1 vial 0.1ml, 4 vials each gene 10^7TU in 1 vial 0.1ml, 4 vials

Box 8 | Vector Maps

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Protocols
Storage: -80oC. Avoid repeated freeze thaw cycles MOI determination 1. 2. 3. Plate target cells to 70-80% conuence in a 96-well plate before infection. Place mWasabi GFP-virus stock (virus titer: 1x10^8 IU/ml) in a 37C water bath and before it totally thaws transfer onto ice immediately. Make a serial dilution of 3-5 different multiplicity of infection (MOI = infectious units/ cell number) to test the target cells. Example 1 x 10^4 TE671 cells in each well of a 96-well plate:
Culture Medium (ul): Polybrene (1mg/ml): Virus (ul): Total volume (ul): 48 1 1 50 47 1 2 50 44 1 5 50 39 1 10 50 29 1 20 50

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two10-cm dishes, or two 6-well or 12-well plates (about 3-4x10^4/cm2). Spray vial with 70% ethanol and wipe dry before placing in tissue culture hood. Gently add 1 ml prewarmed feeder cell medium (alphaMEM or DMEM/F12 with 10% FBS), mix with contents of cryovial and transfer into 15 ml conical tube containing 4 ml prewarmed feeder cell medium. Centrifuge the cells at 200g at room temperature for 5 min and discard the supernatant. Resuspend the feeder cells in 12 ml feeder cell medium. If using a 12well plate: add 0.5 ml feeder cell suspension to each well of 12-well plate containing 1 ml fresh feeder cell media per well. Gently shake the dish left/right and up/ down 10-20 times without swirling the plate to evenly distribute the cells across the plate. Incubate the cells in 37oC, 5% CO2, overnight. CRITICAL STEP When moving the feeder cell plates from the tissue culture hood to incubator, do not swirl the medium, as this tends to cause the cells to accumulate in the center. Immediately after placing the plates in the incubator, slide the plates forward and backward (23 cm) two times, then left to right (23 cm) two times to ensure equal distribution of the cells. Use within 57 days.

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Tilt the plate gently every 30 min. After 3hrs, add 100ul fresh medium. After 48-72 hrs, evaluate mWasabi GFP expression by using uorescence microscope or ow cytometry. Choose the most efcient MOI with the least toxicity for iPS cell derivation.

Induction procedure 1. 2. 3. Plate cells to 70-80% conuence in 12 or 24-well plate before infection Place virus stock in 37C water bath and before it completely thaws transfer onto ice immediately. The optimal MOI to be used depends on cell types and growing conditions. Unnecessarily high viral load will cause toxicity to cells. Try to start with 1-6 ul for each cell type and change the volumes of the remaining materials shown in the MOI test table accordingly (above). Change to fresh medium after two days until ~ day 6. If iPS cell colonies have already appeared, or if the cells have become over-crowded, split and transfer the infected cells to irradiated feeder cells. Some cells may require 2-3 weeks before cell morphology changes. After one day, change culture medium to ES medium (e.g. DMEM + 20% knockout serum replacement, 100ng/ml bFGF, if using bFGF-expressing feeder cells, there is no need to add recombinant bFGF). Change to fresh ES medium every two days, colony formation can be seen in additional 1~2 weeks.

Splitting iPS cells: 1. Split stem cells (~2.5 x 10^5 to 5 x 10^5 cells, or ~10% conuence) into plate with feeder cells: aspirate medium from ESC or iPSC, wash with PBS and add 0.5 ml of 0.05% trypsin. Incubate at 37oC, 5% CO2, for 5 min. Inactivate trypsin with 3 ml stem cell medium (e.g. DMEM + 20% knockout serum replacement, 100ng/ml bFGF, if using bFGF-expressing feeder cells, there is no need to add recombinant bFGF), and collect cell clumps in 15-ml conical tube avoiding making single cell suspension because ESC tends to die in single cell form. Centrifuge at 200g at room temperature for 4 min. Aspirate feeder medium from feeder plates, rinse with one ml of stem cell medium and add 5 ml of stem cell medium and return to incubator. Aspirate and discard supernatant from the conical tube in Step 3, resuspend cells in 5 ml stem cell medium, gently dispense the cell pellet three times, add to feeder cell wells or dishes. Incubate stem cells grown on feeder cells at 37C, 5% CO2, for 48 h. Aspirate medium and replace with stem cell medium every day; if iPSC colony number is low, replace medium every two days.

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Preparing feeder cells: 1. Thaw one vial of irradiated feeder cells by swirling gently in 37oC water bath until all of the contents are thawed. One vial of 2x10^6 cells is sufcient to prepare

6. 7.

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Related Products
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Custom Viral Packaging Service

A. Price Comparation on Typical Items from Competitors
Compare the titer, delivered volume of viral stocks, and prices per million particles from Allele Biotech with other companies which can be found using google to search, and see for
Cat# Allele Biotech GS Company SB Company BC Company ABP-CS-MBRV001 ABP-CS-MBRV002 # CS965A-1 LV002-b-ABM Price $1,499.00 $1,399.00 $2,500.00 $2,047.50 1,250.00

yourself how much value Allele Biotech provides and how little reason you have to prepare your own viruses.

Titer ~1x10^8/ml ~1x10^8/ml ~1x10^8/ml ~1x10^8/ml ~1x10^8/ml

Volume 2ml 2ml 1ml >150ul 1ml

Particles ~2x10^8 ~2x10^8 ~1x10^8 ~1.5x10^7 ~1x10^8

Unit Price (per 10^6) $7.50 $7.00 $25.00 $136.50 12.50

Subcloning Included Not Included Included Not Included Not Included

B. Essential Virus Grant Program

cientic publications report turning a dormant gene into an active one in terms of research activities and this results in signicant demand for cDNA clones, antibodies, expression vectors, etc., all in a short order. Providing reagents for the expansion of research on such genes is scientically 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 such hot genes, (e.g. iPS factors, light-activated ion channels, factors that induce neuronal cells from other cell types). Allele Biotech has custom-package high titer lentivirus and retrovirus using unique

technologies, as described under Services by Category> Viral Packaging. Under this program, Allele Biotech will accept custom viral packaging orders, including cDNAs of or shRNAs against recently established critical mammalian factors. We will evaluate your one page application that explains why the factor should be highly demanded by other researchers. If the application is accepted, Allele Biotech will waive the charges for the project. Instead, it will consider it as an R&D effort and provide shelf product of the same at much lower cost than custom projects. Send in your application with your order today, and our accounting department will promptly forward your request to our review group.

Allele Biotech proudly provide the worlds most powerful viral packaging platform. Introducing cost effectiveness to your research is just the beginning. Imagine how our ingenuity and innovation can pave the way for your research.

Allele Biotech-Introducing Cost Effectiveness to Research