CytoTune™ –iPS Reprogramming Kit

Launching June 15, 2011

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Invitrogen™ CytoTune™ -iPS Reprogramming Kit

What is it?Sendai virus particles used to reprogram somatic cells to induced pluripotent stem cells (iPSCs)

What’s in the kit?4 genes included - all must be used together

• Oct4, Sox2, Klf4, c-Myc (Yamanaka factors)• High titer (3-9e7 CIU/mL), 100uL each

Key Points:1. Up to 100-fold higher efficiency of

reprogramming compared to Lentivirus, Retro, etc.

2. Zero footprint3. Easy to use

Cat. No. Unit Size

A1378001 1 pack (1 of each gene)

A1378002 3 pack (3 of each gene)

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What is Sendai Virus?

● Background

− A respiratory virus of mouse and rat, belonging to the

Paramyxoviridae family, classified as mouse parainfluenza virus

type I.

− Genome of sendai virus is RNA (minus sense).

> No possibility of altering host chromosomes.

− Replicates exclusively in the cytoplasm.

− Non-pathogenic to humans.

● Sendai Virus Vectors

− No genotoxicity: there is no chance of vector integration into

chromosomes of the target cells.

− Wide range of targets: capable of transducing a wide range of

animal cells (including avian and mammalian cells) in both

proliferative and quiescent states.

− High transduction efficiency with low multiplicity of infection (MOI).

− Short contact time of virus with target cells is sufficient to establish

transduction.

− High level of expression of the transgenes.

− Fast expression of the transgenes: expression is detectable as

early as 6-10 hours after transduction, with maximum expression

detected more than 24 hours after transduction.

− Zero footprint: the vectors and transgenes can be eliminated from

the cells.

− No production of infectious particles by the transduced cells.

− Derived from a virus that is non-pathogenic to humans.

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Technical Analysis –Advantages of Sendai Virus

1. Up to 100-fold higher reprogramming efficiency than lentiviral methods2. Sendai is a non integrating virus, and the number of non-iPS colonies are

significantly reduced (Low background)3. Sendai virus can infect a wide variety of cell types4. Only one round of transduction is required for most cell types

Sendaivirus

Lentivirus/ Retrovirus

Adenovirus Episomal/ Minicircle

Protein Modified mRNAs

Efficiency 0.01-1% 0.001-0.01% 0.0001% 0.0001% 0.00001% >1%

Integration No Yes No (DNA)No (DNA)

No No

MultipleTransductions

No No No No/Yes Yes Yes

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CytoTune™ Reprogramming Timeline

Day 1Plate fibroblasts in fibroblast culture medium (DMEM + 10% FBS) into two wells of a 6-well plate to achieve 5x105 cells per well on Day 3.

Day 3 Add CytoTune™ -iPS Reprogramming Kit to your cells.

Day 4-10Replace the fibroblast culture medium (DMEM + 10% FBS) with fresh fibroblast culture medium on Day 4, and then every other day thereafter.

Day 10Harvest cells with trypsin, then plate the cells onto MEF feeder coated plates in fibroblast culture medium.

Day 11Replace the fibroblast medium with complete KnockOut™ Serum Replacement medium.

Day 12-28Observe the cells and replace the spent medium daily. Look for the emergence of cell clumps indicative of iPSCs.

3-4 weeks post transduction

Perform live staining with Tra1-60 or Tra1-81 to select reprogrammed colonies. Manually pick colonies and transfer them to fresh MEF plates.

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Retrovirus vs. Sendai virus:Tra1-81 stain

Phase + Tra1-81Tra1-81

Phase + Tra1-81Tra1-81

Retrovirus –high background, hard to distinguish iPSC colonies.

Sendai virus

Sendai – low background, easier to distinguish iPSC colonies.

Retro-virus

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iPSCs generated using CytoTune™ -iPS Reprogramming Kit

Alkaline phosphatase stain of HDFn cells (BJ strain) 4 weeks after transduction with CytoTune™-iPS

Reprogramming Kit.

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iPSCs generated using CytoTune™ -iPS Reprogramming Kit

Fibroblasts before transduction 3 weeks post transduction

4 weeks post transduction After manual picking and expansion

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Integration Free iPSCs

RT-PCR results of ten iPSC lines generated using the CytoTune™ kit

show the absence of the Sendai virus (SeV) after multiple passages.

Immunocytochemistry with anti-Sendai virus antibodies shows the

absence of sendai virus.

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Technical Analysis

Three reprogramming experiments were performed on BJ (fibroblasts), and obtained efficiency between 0.01-0.1% by Tra1-81 staining

Expression of Pluripotent Markers

EBs differentiate to three germ layers

AFPBeta-III

Tubulin

SMA

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Citations

Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome.Fusaki N et al. Proc Jpn Acad Ser B Phys Biol Sci. 2009;85(8):348-62

Generation of induced pluripotent stem cells from human terminally differentiated circulating T cells. Seki T et. Al. Cell Stem Cell. 2010 Jul 2;7(1):11-4.

For more information, visit www.invitrogen.com/cytotune

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

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