How do you produce your iPS cells?

From AlleleForum: First off, thank you for choosing Allele Biotech for your iPSC experiment needs. Now onto your questions

You asked Q1: How many human fibroblast cells you normally to start for transfection. I understand you use 12-well plate? How many days you wait till the cells grow confluent? If the cells never grow confluent, should I still transfer them to feeder plate? Is it critical for the cells to reach confluent, if it is, could you suggest the reasons to me

We usually plate at 70% or about 10e4-10e5 cells and transduce the cells for 2-3 days. It should become confluent in 2-3 days. There is no need for the cells to become confluent before splitting onto feeder cells. Please note for primary cells, do not wait for the cells to get too confluent because contact inhibition may induce growth senescence before cells are reprogrammed.

Q2: How many cells you plate on the feeder plate, let’s say it is 6-well plate, and how many clones would normally pup out from each well?

From one well of a 12 well plate, you can plate 1/5 onto a well of a 6 well feeder cell plate. From there, you should get plenty of colonies.

Q3: At the time when you need to cut the Loxp sites, what passage number you do, do you have to dispense the iPS into single cell? Do you have a detailed protocol for that? Other than virus, do you have any other means to do the job, like plasmid?

Never dispense iPSC into single cells. They do not grow back well if split into single cells. iPSC colonies should be passaged in patches of cells. To excise loxP, the suggested timing is after 12-14 days when the cells are reprogrammed into iPSC colonies. Just transduce the iPSC colonies with Cre virus.

Q4: Is it true, that the 4-in-1 is more powerful than individual ones? Do you have the construct(4-in-one) for sale?

The 4-in-1 is somewhat more effective than 4 individual ones. For license issues, we do not distribute the construct to customers because we only offer packaging service. Similar type of plasmid DNAs may be accessible from other sources.

If you have any other questions or concerns, please let us know. Thanks again.

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Tags: 4-in-1, feeder cells, iPS, iPS colonies, iPS medium, iPS protocol, lentivirus, loxP Cre, splitting iPS cells

From iPSC to induced beta-cells, iN and iCM: dedifferentiation vs direct reprogramming

The success of inducing pluripotency in primary fibroblasts and other cells with a combination of only a small number of transcription factors suggested that fully differentiated cells might change fate following similar treatments. Since the demonstration of induced pluripotent stem cells (iPSCs), at least three examples have been published where 3 cell type-specific factors were selected from a pool of 10-20 candidates that, when expressed from viral vectors, could induce beta-cells, neurons, or cardiomyocytes.

Induced beta-cells [1]: Ngn3, Pdx1, and Mafa, adenovirus injected to in vivo targets

Induced neurons (iN) [2]: Ascl1, Brn2, and Myt1l, lentivirus infecting mouse embryonic fibroblasts (MEF) or tail tip fibroblasts (TTF)

Induced cardiomyocytes (iCM) [3]: Gata4, Mef2c, and Tbx5, lentivirus infecting cardiac fibroblasts or TTF

In all 3 cases, the change of fate seemed to be via direct conversion, without passing through a progenitor cell fate before further differentiation. Like iPSC reprogramming, direct reprogramming also requires a transient supply of inducing factors. Unlike generating iPSCs, the percentage of cells getting reprogrammed is much higher in direct reprogramming, ~20% in the cases of iN and iCM vs 0.1-1% in iPSC. It is likely that a transient, inductive expression of essential factors jump-starts endogenous factors to establish cell fate specific programs; it has also been illustrated that chromatin remodeling through DNA methylation, histone modifications, etc. accompanies the direct reprogramming events.

The requirement of the full complement of inducting factors may vary depending on how close the original cell type is to the new cell type. iPSCs are typically created by using 4 genes, but can be created with just Sox2, Oct3/4 particularly when the cells to be reprogrammed are less differentiated, such as tissue progenitor cells. Instead of a more “complete” direct reprogramming from unrelated cells to iN and iCM, the induced beta-cells come from exocrine cells, which share parental cells with beta-cells.

Looking into the near future, it should be expected that cell type-specific gene expression profiles are being re-examined or created right this moment to look for candidate gene pools specific to other cell types, starting from those with cell therapy relevance. Lentivirus, retrovirus, adenovirus, or baculovirus for mammalian expression are being constructed to carry them into fibroblasts or cells that are close to the end product of direct reprogramming. In a few months, many of these inducing gene-expressing viruses will become shelf products as high titer viruses from suppliers like Allele Biotech, incorporating tools in viral packaging, fluorescent proteins, and polycistronic gene expression systems.

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1. Zhou, Q., J. Brown, A. Kanarek, J. Rajagopal, and D.A. Melton, In vivo reprogramming of adult pancreatic exocrine cells to beta-cells. Nature, 2008. 455(7213): p. 627-32.
2. Vierbuchen, T., A. Ostermeier, Z.P. Pang, Y. Kokubu, T.C. Sudhof, and M. Wernig, Direct conversion of fibroblasts to functional neurons by defined factors. Nature. 463(7284): p. 1035-41.
3. Ieda, M., J.D. Fu, P. Delgado-Olguin, V. Vedantham, Y. Hayashi, B.G. Bruneau, and D. Srivastava, Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell. 142(3): p. 375-86.

Tags: direct reprogramming factors, iCM, iN, induced beta-cells, induced cardiomyocytes, induced neurons, iPS, iPS lentivirus, iPSCs, MEF, TTF

Do You Know How Well Your Sunscreen Works?

Skin diseases caused by sun exposure include melanoma, basal cell carcinoma, squamous cell carcinoma, photoaging, as well as sunburn and many other conditions. According to the Skin Cancer Foundation, skin cancer is the most common type of cancer in the US. The vast majority of mutations found in melanoma, according to a 2009 study published in Nature [1], are caused by UV radiation.

Currently, commercial sunscreens are composed of physical sunblocks including zinc oxide and titanium dioxide, and chemical UV (ultraviolet lights) absorbers/filters such as octinoxate for UVB and benzophenone for UVA. The compositions of commercial sunscreen products are disclosed by the manufacturer and regulated by the health product regulatory authorities such the FDA in the US. The UV absorbers/filters are organic chemicals that absorb UV lights within a very limited range of wavelength. Consequently, a combination of different chemicals is needed to achieve “broad-spectrum” protection.

Currently the FDA required test of effectiveness of UV protection measures only UVB, which means there is no way of knowing how effective a sunscreen product is against cancer-causing UVA and damaging visible lights [2]. Even though the life style changes in recent time result in more damaging light exposure such as extended sun bathing on beach or tanning in beauty saloons, etc., only 3 new sunscreen active components (and none of new chemical class) have been introduced to the US market in more than 3 decades. There seems to be a gap between the need and the effort for developing substantially improved skin protection products.

1. Pleasance, E.D., R.K. Cheetham, P.J. Stephens, D.J. McBride, S.J. Humphray, C.D. Greenman, I. Varela, M.L. Lin, G.R. Ordonez, G.R. Bignell, K. Ye, J. Alipaz, M.J. Bauer, D. Beare, A. Butler, R.J. Carter, L. Chen, A.J. Cox, S. Edkins, P.I. Kokko-Gonzales, N.A. Gormley, R.J. Grocock, C.D. Haudenschild, M.M. Hims, T. James, M. Jia, Z. Kingsbury, C. Leroy, J. Marshall, A. Menzies, L.J. Mudie, Z. Ning, T. Royce, O.B. Schulz-Trieglaff, A. Spiridou, L.A. Stebbings, L. Szajkowski, J. Teague, D. Williamson, L. Chin, M.T. Ross, P.J. Campbell, D.R. Bentley, P.A. Futreal, and M.R. Stratton, A comprehensive catalogue of somatic mutations from a human cancer genome. Nature. 463(7278): p. 191-6.
2. Botta, C., C. Di Giorgio, A.S. Sabatier, and M. De Meo, Genotoxicity of visible light (400-800 nm) and photoprotection assessment of ectoin, L-ergothioneine and mannitol and four sunscreens. J Photochem Photobiol B, 2008. 91(1): p. 24-34.

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Tags: fluorescence, light exposure, melanoma, skin cancer, skin care, spectra, spectrum, sunburn, sunscreen, UVA, UVB, visible light

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.

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

Allele’s pallet of the super star fluorescent proteins

“Photoblog”–just some fun pictures from our notebooks.

The brightest cyan, green fluorescent proteins, and the brightest ever FP in LanYFP!

These fluorescent proteins are representatives of the growing family or high quality, new generation FPs engineered to enable experiment previously deemed impossible.

Cells infected with lentivirus carrying mWasabi. Lentivirus carrying LanYFP will make most cells much more brighter than this.

The brightest green fluorescent protein with excellent photostability, carried on 10e8 TU/ml high titer lentivirus.

The LanFPs express well in bacteria.

Project planning is under way to test the cytotoxicity of lanFPs in different mammalian cell lines and in vivo with a focus on neurons.

The FPs fold so strongly that they fluorescence even in SDS-PAGE.

FPs in SDS PAGE–a closer look

FPs in gel cassette over UV lights

FPs in gel cassette under blue LED

The purified FPs can be used as “real time” protein markers.

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Tags: Fluorescent proteins, FP, FPs, FRET, GFP, lab note, lentivirus, mCherry, mTFP1, mWasabi, photos, pictures