Allele Biotechnology Announces New advance in production of human stem cells

This week in the journal Scientific Reports (Nature Publishing Group) scientists from Allele Biotechnology describe an important advance in the generation of stem cells capable of producing all the different tissues of the human body. In an article entitled “Feeder-Free Derivation of Human Induced Pluripotent Stem Cells with Messenger RNA,” Allele’s scientists present the fastest and safest method yet for converting ordinary human skin cells into “induced pluripotent stem cells” (iPSCs).

The scientific efforts were led by Dr. Luigi Warren, whose pioneering work on “footprint-free” reprogramming using messenger RNA was the foundation for Allele’s breakthrough. Through the united efforts of Dr. Warren and the scientists at Allele Biotechnology, his technique was re-engineered to increase cell conversion efficiency and eliminate any use of potentially unsafe reagents, while substantially reducing the time and effort needed to make stem cells. Dr. Warren believes that because of its advantages this technology “should become the method of choice for iPSC cell banking.”

According to Dr. Jiwu Wang, corresponding author on the paper and CEO of Allele Biotechnology, “This advance in stem cell derivation will enable both fundamental scientific research and clinical applications which has been the mission of Allele Biotechnology from its inception.”

Allele Biotechnology and Pharmaceuticals Inc. is a San Diego-based biotechnology company that was established in 1999 by Dr. Jiwu Wang and colleagues. A research based company specializing in the fields of RNAi, stem cells, viral expression, camelid antibodies and fluorescent proteins; Allele Biotechnology has always striven to offer products and services at the cutting edge of research.

Allele Biotechnology and Pharmaceuticals Inc.
Jiwu Wang, Ph.D., 858-587-6645 Ext 3
President and CEO
iPS@allelebiotech.com
fax: 858-587-6692
www.allelebiotech.com
Press release by BusinessWire. Also see Yahoo!News, Reuters, The Herald, etc.

Tags: flourescent proteins, human fibroblasts, iPS, iPSC, iPSCs, Lin28, MO3, mRNA reprogrammin, Myc, Nanog, Oct4, reprogrammin, Sox2, stem cells

Cord Banking and iPS Cells

Umbilical Cord Banking (UCB) has been a popular discussion topic in the United States since the first Cord Bank was established in New York in 1992. Since the first cord blood transplantation in 1988, there have been over 780,000 UCB donations to private banks and 400,000 UCB donations to public blood banks worldwide. There has been such a great number of donations because UCB is full of hematopoietic progenitor cells, which makes it a more desirable solution to genetic, metabolic and immune disorders, over bone marrow and blood. Because of the nature of UCB, the recipient does not need to be an immunological match, there is a lower rate of infection and it is much easier to acquire than bone marrow, making it the ideal form of treatment for many patients and practitioners.

Over twenty years later, a new technology is emerging that could provide some clarity to the “to donate or not to donate” debate: induced pluripotent stem cells (iPSC). Derived from adult cells, iPSCs have the potential to be used like UCB or reprogrammed into specific tissue like myocytes. This potential opens up banking to countless individuals born before 1992, who never had an option to bank their UCB. With this unbounded potential, should iPSCs be banked liked UCB? Supporters argue that there has been enough evidence thus far to start a bank, however, most people seem to agree that too much is unknown about iPSCs and their use in humans. With that, most are in agreement that iPSC research is absolutely needed so banking can become a reality in the future.

For now iPSCs will remain in the testing and research phase, however, based on current research, iPSCs have the potential to enhance Cord Blood that has already been banked, perhaps providing some relief to public banks in the future (Broxmeyer, 2010). Though the potential of iPSCs is endless, more work has to be done before they are placed in humans and considered a viable banking system.

Tags: Cord Blood, ESCs, iPS, Stem Cell Banking, stem cells

Making Transfection-Grade mRNA by IVT (In Vitro Transcription)

RNases are an often feared in molecular biology labs because of their high stability and ominous presence in virtually all living systems. Consequently, people who work with RNA are trained to exercise extreme caution to avoid RNA degradation: change gloves often because human hands ooze RNases; use only sterilized labware as microbes may be sources of RNases; for surfaces that can’t be autoclaved, use sprays like “RNase Zap” (SDS- or guanidine-containing solutions). Such cautionary steps are especially necessary when dealing with low abundance RNA samples.

RNAs can be produced by in vitro transcription (IVT), a simple reaction requiring only a DNA template (double-stranded or even single-stranded DNA as long as the promoter region is double-stranded), RNA polymerase (from T7, SP6, or T3 phage), NTPs, and a reaction buffer that provides appropriate salt and pH. Standard NTPs may be replaced with modified ones to either increase stability or to reduce immune-response when transfected into cultured cells. Additionally, a 5’ cap structure may be added during IVT for further stabilizing mRNAs inside the cells post transfection. Using a commercially assembled kit, one can routinely produce 40-50 µg of mRNA from 1 µg of DNA template in a single 20-50 µl reaction.

At such high concentrations, IVT mRNAs are not nearly as sensitive to RNase-mediated degradation as low-abundance samples. The mRNA can be easily observed on agarose gels that are regularly used for DNA, and their integrity can be monitored after transcription or storage. In most cases one distinct band of mRNA from an IVT reaction is obtained as long as a clean DNA template is used. Preparing a good, uniform IVT template is critical to prevent aberrant products. By using high quality templates, IVT mRNA produced in your own lab are often higher in quality than mRNAs purchased from current commercial sources (Figure in Blog shows mRNAs generated by IVT for R-iPSC). Sometimes there are minor bands created during IVT, but they normally do not interfere with the intended uses of the mRNA, and can be purified away with a purification kit (by using a discriminating purification scheme such as Allele Biotech’s Surface Bind RNA Purification, smaller species can be specifically removed, a separate topic for another blog).

Once produced, mRNAs can be stored at -20C for months, or -80C nearly indefinitely.

mRNAs generated by IVT for R-iPSC

Tags: fluorescent protein, iPS, IVT, luciferase, mRNA, nuclease, recombinase, RiPSCs, RNA degradation, RNase, TALEN

Year-end message from Allele Biotech

The year 2011 has been an exciting and eventful year for many people. Throughout the year, we have been working diligently to bring the best research methods in many areas to our fellow researchers through innovation and entrepreneurship. Thanks in part to the government’s stimulus and grant support in 2011, we established several new product lines, including the Stealth iPS induction mRNA templates and reagents, a great new photoconvertible fluorescent protein in mClavGR2 (through collaboration with academic colleagues), and a highly efficient lentivirus-based shRNA packaging service as a result of an NCI SBIR contract.

As you all must have noticed by now, in July we redesigned our website to present our products in an easier, more user friendly manner, while adding a convenient online purchasing system. We have received a lot of positive feedback from customers telling us how “cool” the new site is, and how easy it is to use and redeem promotions. Towards the end of the year, our dedicated marketing and sales teams reinstated our biweekly email newsletters (to receive our messages on new discoveries and technologies, or be the first to use our promotions, sign up online under “Newsletter”).

All of these efforts would have been meaningless without our customers, who ultimately gave us the opportunity to be in the business we love and are trained to do. By selecting our products, sending us feedback, and “retweeting” or “reposting” our messages, you have been tremendously valuable to every one of us here at Allele. We thank you from the bottom of our hearts. In return, we will continue to invest and do our very best to provide new tools for advancing your research. Watch for our brand new monomeric fluorescent protein that can be nearly 10 times brighter than EGFP; a more powerful iPSC generation method that could potentially reprogram in just a few days, and much much more in 2012!

Tags: 2012, EGFP, iPS, iPSC, mRNA templates, NCI SBIR, new fluorescent proteins, reposting, retweeting

Top 10 List of Most Viewed AlleleBlogs in 2011

The ballot is in—among the “usual suspect” hot topics, iPS takes the top honor and most entries; Camelid antibodies, although not really presented as a typical AlleleBlog in 2011, made it to the top 3. shRNA cloning and RNAi screening are still on a lot of people’s minds, so it seems.

Method: total visits to each blog since our new webpage was launched in July was counted.

1) Fusion of the Transcription Domain to iPS Factors Radically Enhances Reprogramming
http://blog.allelebiotech.com/2011/10/fusion-of-the-transcription-domain-to-ips-factors-radically-enhances-reprogramming/

2) Methods of iPSC Generation Update
http://blog.allelebiotech.com/2011/08/methods-of-ipsc-generation-update/

3) About 50 Papers Cited the Use of GFP-Trap Camelid Antibody So Far in 2011
http://blog.allelebiotech.com/2011/09/about-50-papers-cited-the-use-of-gfp-trap-camelid-antibody-so-far-in-2011/

4) Big Potential in Using Protozoans for Producing Mammalian Proteins
http://blog.allelebiotech.com/2011/09/big-potential-in-using-protozoans-for-producing-mammalian-proteins/

5) How do you make shRNA-expressing viruses for function screening?
http://blog.allelebiotech.com/2011/11/how-do-you-make-shrna-expressing-viruses-for-function-screening/

6) Creating ground-state human iPSCs
http://blog.allelebiotech.com/2011/10/creating-ground-state-human-ipsc/

7) Recombinase-Mediated Cassette Exchange (RMCE) and Integrase Swappable in vivo Targeting Element (InSITE)
http://blog.allelebiotech.com/2011/03/recombinase-mediated-cassette-exchange-rmce-and-integrase-swappable-in-vivo-targeting-element-insite/

8) Development of Cell Lines from iPSCs for Bioassays
http://blog.allelebiotech.com/2011/11/development-of-cell-lines-from-ipscs-for-bioassays/

9) Choosing siRNA, shRNA, and miRNA for Gene Silencing
blog.allelebiotech.com/2010/02/choosing-sirna-shrna-and-mirna-for-gene-silencing/

10) Allele Biotech’s Box Swap Program
http://blog.allelebiotech.com/2009/07/allele-biotechs-box-swap-program/

Have a successful 2012!

Tags: camelid antibodies, environment friendly research, GFP-Trap, iPS, mRNA iPS, RiPSC, RNAi screening, shRNA lentivirus packaging