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

How do you make shRNA-expressing viruses for function screening?

Most people use standard cloning procedures when trying to insert shRNA templates into lentiviral vectors, i.e. anneal a pair of long oligos with sticky ends and ligate the dsDNA into a linearized plasmid with compatible overhangs. However, since typical lentiviral vector plasmids have terminal repeats and are relatively large, when ligated to hairpin sequence-containing shRNA templates, recombination often occurs inside bacteria that results in smaller plasmids. This problem is common for cloning shRNA or other unstable DNA pieces into viral vectors. This cloning issue is further compounded by the fact that it is difficult to sequence any shRNA template region because the hairpin may block the progress of the DNA polymerase used in sequencing, sometimes requiring several repeats under different sequencing conditions, incurring high costs charged by sequencing service providers.

To deal with these aspects of the cloning difficulties, particularly for the purpose of increasing cloning efficiency RNAi-based screening, we compared three different strategies

First, we built a smaller shRNA cloning vector to clone and sequence shRNA templates prior to transferring to lentiviral vectors. This smaller vector does not have a severe recombination problem and is easier to sequence in the hairpin-containing region. After an initial round of cloning with this new vector, we further improved it by inserting an XbaI and a NheI site between the BamHI and SpeI insertion sites, so that any plasmid preparations can be screened for recombinants by a simple XbaI or NheI digest before sequencing. After cloning into this intermediate vector, the shRNA expression cassette can be transferred into the lentivirus vectors with some flanking viral sequences so that the insert size will be around 1kb.

Second, we developed a novel DNA preparation procedure after realizing that DNA damage during miniprep of vector plasmids and gel purification of vector fragment increased recombination of these constructs, which were already less stable than usual due to hairpin structures. This procedure of DNA preparation avoids UV or guanidium exposure, which can cause nicks on double-stranded DNA and facilitate recombination. This new procedure relies on purifying DNA through surface-binding to regular reaction tubes treated with a proprietary reagent (SurfaceBind Purification). The process simply requires adding a proprietary, guanidium-free binding buffer to the DNA, which has been processed in a specially coated tube (eppendorf or thin-wall PCR tube), and purifying directly in the same tube. Vectors prepared this way indeed provide more colony counts and a higher percentage of correct constructs as shown by our test runs. The procedure also requires less time and the purified DNA can be dissolved in volumes as small as a few microliters.

Third, to enable truly high throughput shRNA screening (i.e. looking for effective RNAi reagents), we further tested and adapted a ligationless cloning protocol that can be handled by a liquid handler almost entirely. In order to increase throughput, we designed a drastically different procedure that could bypass ligation and sequencing altogether before functional tests. Briefly, DNA molecules that would provide enhanced recombination were created by one round of PCR, purified directly in the surface bind PCR reaction tubes (any template DNA would be removed with DpnI enzyme that cuts non-PCR DNA), pooled, and transformed in bacteria directly. DNA plasmids from transformed bacteria can be used for lentivirus packaging, bypassing sequencing at the initial screening stage, and choose single colonies for sequencing only after a shRNA sequence shows promise in functional assays. This is based on the fact that such cloning rarely has any background colonies, and that among all oligos (if using the correct grade of oligos from validated suppliers) inserted this way, a good portion encodes the correct sequence.

New Products of the week: 100x 15mm EcoCulture Vented Dishes for better stem cell attachment and less plastic waste to the environment, APB-CS-114TC.

Promotion of the week: Buy 1 Stealth Express IPS Induction PCR Template Set, get 1 SurfaceBind RNA Purification Kit free. Use code FreePureRNA.

Tags: lentivirus packaging, lentiviurs shRNA, RNAi, RNAi screening, shRNA, shRNA cloning, shRNA lentivirus packaging, shRNA library

Development of Cell Lines from iPSCs for Bioassays

The reprogramming of differentiated somatic cells to pluripotency holds great promise for drug discovery and developmental biology. Using immortalized cell lines for drug screening assays has its limitations, such as questionable relevance; and the use of primary cells is often hindered by supply difficulties. Thanks to pioneering work by the Yamanaka, Thompson, and other groups, the feasibility of creating iPSCs has generated an opportunity to provide cell lines with stem cell properties in a virtually unlimited supply [1, 2]. These cells can be derived into different cell types for specific assays, even with patient- or genotype-specific background. Technologies are being developed to produce re-differentiated cells of a number of lineages.

Take cardiomyocytes as an example. There are a number of conventional methods for inducing stem cells into cardiomyocytes: through embryoid body (EB) formation, co-culturing with visceral endoderm-like cell line (END-2), and monolayer caridomyocyte differentiation with defined growth medium and protein factors [3]. A recent publication showed that using appropriate concentrations of BMP4 and activin-A in BSA-containing medium cardiomyocytes might be achieved from iPSCs or ESCs in about 6 days [4].

Transdifferentiation, or direct reprogramming, by introducing a group of 3 cardiomyocyte-specific factors, investigators could directly program cardiac or dermal fibroblasts into cardiomyocyte-like cells [5]. Although much refinement and characterization of these directly reprogrammed cardiomyocyte-like cells, termed iCMs, will be needed before the process can become widely used, this work raised the possibility of quicker and perhaps more efficient ways of generating cells for assays. Similar transdifferentiation has resulted in induced neuron (iN) cells, also by introducing 3 tissue-specific transcription factors [6]. Therefore, it seems that by using defined combinations of tissue-specific transcription factors it is possible to generate cells of different tissue types. It is also possible that by using different, developmental stage-specific transcription activator sets, transdifferentiation can be conducted in a stepwise way and make sure cells at each step is pure. This strategy may be particularly attractive if its efficiency can be improved by the techniques developed for iPSC creation. After all, reprogramming to pluripotency and transdifferentiation to different tissue types must share certain mechanistic steps in their respective processes.

In addition, it has been reported that by briefly overexpressing the Yamanaka iPS factors and controlling growth conditions, mouse fibroblasts could be transdifferentiated up to 40% in 18 days without reversing back to pluripotency [7]. It would be interesting to see if by transient expression of iPS factors via mRNA then switching to cardiomyocyte-specific transcription factors, we can increase the efficiency for direct reprogramming. Use of chromatin-modifying chemicals that were already shown to directly reverse and alter cell fates might also be used to assist direct reprogramming. We believe that a systematic approach for studying these reprogramming aspects should benefit the iPS fields.

1. Takahashi, K. and S. Yamanaka, Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006. 126(4): p. 663-76.
2. Yu, J., et al., Induced pluripotent stem cell lines derived from human somatic cells. Science, 2007. 318(5858): p. 1917-20.
3. Vidarsson, H., J. Hyllner, and P. Sartipy, Differentiation of human embryonic stem cells to cardiomyocytes for in vitro and in vivo applications. Stem Cell Rev, 2010. 6(1): p. 108-20.
4. Elliott, D.A., et al., NKX2-5(eGFP/w) hESCs for isolation of human cardiac progenitors and cardiomyocytes. Nat Methods, 2011.
5. Ieda, M., et al., Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell, 2010. 142(3): p. 375-86.
6. Pang, Z.P., et al., Induction of human neuronal cells by defined transcription factors. Nature, 2011. 476(7359): p. 220-3.
7. Efe, J.A., et al., Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy. Nat Cell Biol, 2011. 13(3): p. 215-22.

New Products of the week: T7 RNA Polymerase, high quality for demanding in vitro transcription requirements.

Promotion of the week: GFP-Trap, buy 2 of any package and get 1 of equal or less value free. Use code FreeTrap, follow deals quickly on Facebook.

Tags: cardiomyocyte, didifferentiation, direct reprogramming, Gata4, iCM, iN, iPSC, Mef2c, redifferentiation, Tbx5, transdifferentiation

Creating ground-state human iPSCs

Murine pluripotent stem cells can exist in two distinct states, blastocyst-derived LIF-dependent embryonic stem cells (ESCs) and epiblast-derived bFGF-dependent stem cells (EpiSCs). Murine ESCs and similar iPSC lines are more of the “ground-state” in terms of developmental status, as reflected by the lack of X chromosome inactivation in female cells and their abilities to pass as single cells. Human iPSCs, like human ES cells, are more similar to mouse EpiSCs. Unfortunately these human pluripotent stem cells are difficult to genetically manipulate, e.g. knockin or knockout. They also grow slowly, with doubling time averaging 36 hours. In order to create ground-state human iPSCs, several approaches have been tested, including reprogramming iPSC-derived fibroblasts, continuously expressing 5 iPS factors (Oct4, Sox2, Nanog, c-Myc, and Klf4), or using chemicals to inhibit chromatin modifying enzyme HDAC. While these approaches succeeded to certain degrees, the resulting cell lines seem to have some limitations, such as limited passage numbers.

Retinoic acid (RA) signaling is involved in many aspects of embryonic development. RA receptor (RAR), together with one of its heterodimerization partners, steroid hormone receptor Lrh-1, was recently found to be able to synergize with the 4 common iPS factors (Oct4, Sox2, Klf4, and c-Myc) to induce mouse and human fibroblasts into ground-state iPSCs. The pluripotent cells created by the so-called F6 factor combination show no X chromosome inactivation if from female origin, can fully activate the endogenous Oct4 promoter, express Rex1 (which is specific to mouse ESCs, not EpiSCs), and grow with a 16 hour doubling time. All these mouse ESC-like features were achieved without detectable expression of the exogenous factors once iPSC colonies formed, indicating transient F6 expression is capable of effectively initiating endogenous stem cell factors. Remarkably, these stem cells can maintain their undifferentiated status in mouse ESC medium for 50 passages or more. This work, published this month in Proceedings of National Academy of Science USA [1], provided the stem cell research and application field with a very desirable choice of human stem cells.

As opposed to ~16 days with F4, it appears that the time required to induce adult fibroblasts into pluripotent stem cells is as short as 4 days if F6 factors are introduced on a murine stem cell virus (MSCV) vector with an integrated piggyback transposon. As the authors noted in their discussion, the speed-up benefit should be particularly advantageous for transient transfection approaches such as mRNA reprogramming. The bottom line from this paper and the engineered factor papers (see the previous AlleleBlog article under “iPS and other Stem Cells”) is that iPSC reprogramming is only going to get faster, which means that hopefully in the near future creating iPSCs will become a routine experiment as easy as a simple transfection.

Wang, W., J. Yang, et al. (2011). “Rapid and efficient reprogramming of somatic cells to induced pluripotent stem cells by retinoic acid receptor gamma and liver receptor homolog 1.” Proc Natl Acad Sci U S A.

New Products of the week: ARCA, modified cap analog for in vitro transcription of mRNA.

Promotion of the week: Friday special this week, 15% off all iPS viral particle products if using code “ViraliPS” when ordering online at allelebiotech.com, by email, or fax.

Tags: EpiSCs, feeder cells, ground state stem cells, HDAC, iPSC, Lrh-1, Lrh1, RAR, RAR heterodimer, stem cell medium, X chromosome inactivatin