iPS cells can be generated by introducing a different set of 4 cDNAs: Oct4, Sox 2, Nanog, and Lin28. In combination with chemicals such as valproic acid (VPA) or RNAi reagent against p53, as few as 2 genes (Oct4 and Sox 2) were used to induce pluripotent stem cells. The original set of inducing genes (Oct4, Sox2, c-Myc, and Klf4) are often chosen to start iPS experiments

Most purchased from Allele:

Lentiviral particles expressing Oct4, Sox2, c-Myc, and Klf4, available in stock, buy now, have iPSCs in 1-2 weeks.

Integrating vectors, such as retrovirus or lentivirus, are often used because of the low efficiency of iPS production. However, random integration of these vectors, together with the oncogenic nature of some of the inducing genes, pose a risk of cancer formation by these iPS cells in human therapy. For this reason, non-integrating methods, such as adenovirus or transient transfection of plasmids capable of episomal expression, were developed. In addition, methods of removing integrated sequences with recombinases or transposases can be used.

Learn More  

Retroviral and Lentiviral vectors for expression of human Oct4, Sox2, c-Myc, Klf2, Nanog, and Lin28.

The retroviral vector plasmid which contains the oriP/EBNA1 system for episomal expression are also available.

Allele´s HIV-based lentiviral vector pLICO (implico, Latin for intertwine) and MMLV-based retroviral vector pCHAC (Chac, Mayan for God of culture and growth) have the options of including an IRES driven mWasabi or U6 promoter driven shRNA cassettes.

Allele offers pre-packaged, titer-determined, high titer lentiviral particles ready to infect target cells, including non-dividing cells.

Advantages:

• High vector stability: non-homologous R regions in the vector backbone.
• High vector safety: extensive deletions including the major HIV splice donor site in the RNA packaging signal and a maximal U3 deletion (US patents 6,207,455, 6,531,123)
• High vector titer: state-of-the-art vector production method

Learn More  

Lentiviral particles expressing shRNA against p53 can be used to dramatically increase the efficiency of iPSC generation, as published in Nature on Aug 9, 2009. Allele shRNA against p53 Lentivirus particles:

• High titer, pre-packaged, titer-determined, ready-to-use
• Puromycin resistant gene or fluorescent protein for selection
• Combined with human telomerase hTERT cDNA for further increasing iPSC production rate, from 2 weeks to 7 days

Irradiated mouse MEF or human fibroblasts that express bFGF to support iPSC growth are provided as ready-to-use vials. Simply thaw and plate as feeder cells. MEF is good for mouse iPSC reprogramming; for human iPSC, human fibroblast feeders are preferred.

• Already irradiated and tested, ready-to-use
• MEF for mouse iPSCs, human fibroblasts for human iPSCs
• Expressing bFGF, no need to supply recombinant bFGF as medium supplement

Learn More  

Mouse embryonic fibroblasts (MEF) expressing murine bFGF and LIF, irradiated for direct plating as feeder cells to support iPSC generation using mouse cells. Leukemia inhibitory factor (LIF) is a 20 kDa protein that is known for its ability to inhibit the differentiation of embryonic stem cells in mice and contribute to stem cell selfrenewal. Human and mouse LIF proteins share 79% sequence homology and exhibit cross-species activity. However, LIF inhibition of stem cell differentiation appears to be mouse-specific.

• Already irradiated and tested, ready-touse
• MEF LIF for maintaining mouse iPSCs
• Expressing bFGF, no need to supply recombinant bFGF as medium supplement

Learn More  

Nanog, Oct4, and Rex1 promoters have been reported to display ES/iPS cell-specific expression under undifferentiated state. Allele Biotech provides all three promoter-fluorescent protein (FP) reporters to confirm the state of the reprogrammed iPSCs. A GFP or RFP gene was cloned behind these ES-specific promoters to offer convenient fluorescence tracking of undifferentiated cells.

Lentiviral infection have advantages over other gene-delivery methods including high-efficiency infection of dividing and non-dividing cells, long-term stable expression of a transgenes, and low immunogenicity.

• High viral titer than other lentiviral vectors
• Reliable expression levels
• Self‐inactivating vectors

Particularly in the fields of iPS cells and RNAi where high efficiency gene transfer is critical, lentiviral vectors are increasingly the gene transfer system of choice for non-dividing primary and other difficult-to-transfect cells. Allele´ HiTiter™ Lentivirus systems (US patent 6,620,595) provide high titer and improved safety due to some of the most advanced and unique features:

• Further removed overlapping sequences between transfer and packaging plasmids
• Enhanced poly(A) signal to increase titer and gene expression, and prevent read-through transcription of neighboring genes
• Improved packaging plasmids to increase packaging efficiency and to reduce the likelihood of recombination between vector components

Retroviruses are considered the easiest and fastest means to deliver genes stably to mammalian cells especially when packaged with the widely-used Phoenix™ system. The system provides either an Ecotropic packaging system (for dividing murine or rat cells) or an Amphotropic system (for dividing cells of most mammalian species, including human). Allele´ pCHAC retroviral vectors share similarities to Moloney Murine Leukemia Virus (MMLV), with the following options:

• mWasabi green fluorescent protein as transfection marker
• Integrated Allele engineered shRNA expression cassette for the highest efficiency of RNAi (US patents 7,294,504 and 7,422,896)