Allele Biotech Spotlight Promo for ASCB Dec 09 Meeting!

This year our President and CEO, Dr. Jiwu Wang Ph.D., will be presenting at the American Society for Cell Biology meeting in San Diego, December 5th through 9th. Dr. Wang will be presenting results of two studies that involved the Allele Biotech Fluorescent Proteins and iPSC product lines:

Monomeric photoconvertable fluorescent protein variants produced by directed evolution for brightness and efficient photoconversion – a collaborative effort with the Campbell lab at the University of Alberta

Increased efficiency and speed of reprogramming of human cells into induced stem cells using high-titer lentiviral vectors encoding cell cycle progression and survival genes – a collaborative effort with the Chang lab at the University of Florida

In honor of this prestigious occasion Allele Biotech is having a Spotlight Promotion on all Fluorescent Protein and iPSC Products! The promotions, which will vary from product to product, will include 10% and 20% off price reductions, FREE shipping, and even “Buy 2 get one Free” deals!

Products eligible for the Spotlight Promotions begin with:

ABP-FP-____ Catalog

ABP-SC-____ Catalog

To qualify for these promotions you must be attending the ASCB meeting in San Diego and provide us with a copy of your registration form or be one of our loyal facebook, twitter, or myspace friends. Any questions can go to oligo@allelebiotech.com

Call for details and ask for info on the Spotlight Promotions! Offers good now through December, 9th 2009!

New Product of the Month 11/23-29/09: ThermoExp500 PCR machine (thermocycler) $4,250.00, with almost twice as fast temperature ramping as MJ’s TC1000, and more reliability.

Tags: Allele Biotech Promotions, Allele Biotech Sales, American Society for Cell Biology, ASCB San Diego 2009, fluorescent proteins discount, free shipping, iPSC discount, MJ research, PCR machine, TC1000, thermocycler

Highly Efficient and Non-Integrating Vectors for Generating iPSC

The Challenge and Potential Impact

The proliferative and developmental potential of human stem cells offer virtually unlimited access to the differentiated cells that make up the human body [1]. Stem cell has become one of fastest moving areas biomedical research has ever seen. Tests at all stages ranging from cell differentiation to animal models to human clinical trials have begun within a very short period of time aiming at treating a host of diseases. The excitement generated by the vast potential of stem cells is not only felt by members of the health research community but also has caused great interest and awareness from the general population. Until recently, embryonic stem (ES) cells, derived from the inner mass of blastocysts, have been used for these studies. The use of human embryos in creating human ES cells, however, faces ethical controversies.

As a major advancement in the stem cell field, it has recently been shown that mouse and human differentiated cells may be reprogrammed into stem-like, pluripotent cells by the introduction of defined transcription factors [2-4]. The availability of induced pluripotent stem cells (iPS cells or iPSCs) could provide an ethically acceptable and relatively easy-to-access alternative to human ES cells. Furthermore, it is anticipated that therapies developed with iPSCs could circumvent the problem of tissue rejection following transplantation in patients by creating patient- or even tissue-specific pluripotent stem cells.

Still, great challenges stand between the current methods for generating iPSCs and their therapeutic potential. The use of integrating viral vectors has limited therapeutic potential due to the increased risk of tumor formation. It is therefore important to develop safe, effective and efficient targeting and delivery systems to produce iPSCs.

Because of this importance, multiple methods have been published within the last year that used delivery systems other than the retroviral or lentiviral vectors employed in the original iPSC publications. However, these newly reported methods have not addressed all of the known difficulties facing iPSCs creation. For instance, very low efficiency of transient transfection of selected cDNAs plasmids into primary cells lowers the already abysmal percentage of adult cells that can be reprogrammed with retroviral vectors [5]. Non-integrating adenovirus has a very short cDNA expression time period and thus requires repeated deliveries [6]. The application of episomal expression element such as oriP/EBNA1 helps sustaining longer expression time, but presently it is carried on a plasmid vector and does not improve transfection efficiency. Transposase and the Cre recombinase were used to remove integrated transgenes after the induction is completed, but the integration nevertheless occurred at multiple sites and requires careful and stringent analysis to make sure the reversion is complete; even so, elements of the vector may remain in the iPSCs genome [7, 8].

We wish to take this challenge and use it as an opportunity to develop a synthetic targeting and delivery system that will have the advantages of safe handling, no integration, prolonged and efficient reprogramming gene expression, and high transduction efficiency into broad cell types.

Baculovirus has been used in mammalian cells for many years (e.g. the BacMam system). Engineered baculovirus expression vectors (EBEV) will be exploited as a carrier for reprogramming genes for deriving induced pluripotent cells (iPSCs) from human adult cells. It has been well established that baculovirus can infect mammalian cells with broad tropism yet are very safe for regular laboratory handling.The elements planned for Allele Biotech’s new iPS generating system will be novel in the following aspects:

a) Promoter and mRNA structures for maximum level of cDNA expression in mammalian cells
b) Extended presence in the nucleus for sustained cDNA expression for weeks
c) Cleavable fluorescent protein for cell tracking and sorting
d) Auxiliary packaging constructs for increasing tropism to infect a broad range of human adult cells

Allele Biotech’s Design of Baculovirus for iPSCs

A) Mammalian Expression: In order to express reprogramming cDNAs in human cells, a mammalian promoter cassette will be inserted into the transfer vector to be used with Allele Biotech’s Sapphire Baculovirus genomic DNA. This system, derived from Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), has been provided commercially for 10 years by our team and proved to be one of the best baculovirus systems because of its ease to use and high efficiency. Heterologous promoters have been used by us and others to create baculoviral vectors with dual or triple promoter for protein expression in insect, mammalian, and even bacterial cells such as Novagen’s pTriEx vectors and Allele’s pMBEVS. For the purpose of this proposal, we will remove insect expression cassette altogether and use a CAG promoter for driving expression solely in human cells.

B) Broader Tropism: Baculoviruses has been used for gene delivery to various hepatic and nonhepatic mammalian cells albeit the transduction of certain nonhepatic human cells is about 10-100 fold less effectively than hepatic cells [9]. Incorporating vesicular stomatitis virus G protein (VSVG) in the viral particle surface can greatly increase their effective transduction in a much broader range of mammalian cells [10]. For pseudotyping baculovirus, we will use a coinfection protocol with a recombinant baculovirus expressing VSVG. We expect that incorporating VSVG will ensure that human skin fibroblasts would be infected efficiently for Aim 1; and that different human cells could also be reprogrammed to become iPSCs via the proposed pathway in Aim 3 (see below). Different proteins or peptides might be tried as backup methods for pseudo-typing should VSVG not provide satisfactory results.

C) Fluorescent Marker: We will insert a fluorescent protein (FP) either expressed on a separate cassette or driven by an IRES downstream of reprogramming genes. This feature will facilitate tracking the infected cells, monitoring transgene expression, and enriching infected cells. Allele Biotech is one of the few suppliers/developers of fluorescent proteins. Our exclusive mTFP1 (cyan) and mWasabi (green) proteins are about 3 times brighter than EGFP and true monomers with great photostability and pH insensitivity, which should make them great choices for EBEV.

D) Promoter: The CAG promoter, which has been shown to be a strong promoter in mammalian cells and preferred for BacMam expression, will also be used in the EBEV vectors. We have previously designed pMBVES, a baculovirus vector for glycoprotein expression in mammalian cells that contains such a CAG enhancer/promoter. The CAG composite promoter also encompasses an exon1-intron-exon2 segment that will help mRNA processing and export to cytoplasm for translation. We will clone this fragmentfrom pMBVES into the proposed EBEV vectors.

E) RNA Elements: The 5’ UTR region on the mRNA will be examined and any hairpin structures with ??G < 30 kcal/mol or even <20 kcal/mol but with >65% GC will be disrupted. This step will ensure that maximum production be achieved at the translational level [11]. Post-transcriptional regulatory element (PRE) will be included to further boost gene expression. It has been shown that Woodchuck hepatitis virus (WPRE) increases transgene expression by many folds for various viral vectors, and there has been at least one case for a BacMam vector [12]. WPRE will be cloned from Allele Biotech’s existing HiTiter Lentiviral Vectors and inserted in the 3’ UTR upstream of the SV40 polyA signal.

F) Episomal Expression: Originally derived from Epstein-Barr virus (EBV), phophoprotein nuclear antigen 1 (EBNA1) ensures that oriP-containing DNA replicate once per S-phase during cell circle and is maintained in the nucleus as episomes. Although mostly applied to plasmid vectors, such as in one of the iPSC reports [13], the oriP/EBNA1 system can be incorporated into baculovirus systems for sustained mammalian expression [14]. Aside from episomal maintenance, oriP/EBNA1 could further up-regulate transcription of adjacent genes [14]. For these reasons, we will clone the oriP sequence together with the EBNA1 expression cassette from Allele Biotech’s Phoenix Retrovirus vector pBMN-GFP to EBEV vector.

Bibliography and Reference Cited
1. Thomson, J.A., J. Itskovitz-Eldor, S.S. Shapiro, M.A. Waknitz, J.J. Swiergiel, V.S. Marshall, and J.M. Jones, Embryonic stem cell lines derived from human blastocysts. Science, 1998. 282(5391): p. 1145-7.
2. 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.
3. Takahashi, K., K. Tanabe, M. Ohnuki, M. Narita, T. Ichisaka, K. Tomoda, and S. Yamanaka, Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 2007. 131(5): p. 861-72.
4. Yu, J., M.A. Vodyanik, K. Smuga-Otto, J. Antosiewicz-Bourget, J.L. Frane, S. Tian, J. Nie, G.A. Jonsdottir, V. Ruotti, R. Stewart, Slukvin, II, and J.A. Thomson, Induced pluripotent stem cell lines derived from human somatic cells. Science, 2007. 318(5858): p. 1917-20.
5. Okita, K., M. Nakagawa, H. Hyenjong, T. Ichisaka, and S. Yamanaka, Generation of mouse induced pluripotent stem cells without viral vectors. Science, 2008. 322(5903): p. 949-53.
6. Stadtfeld, M., N. Maherali, D.T. Breault, and K. Hochedlinger, Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse. Cell Stem Cell, 2008. 2(3): p. 230-40.
7. Woltjen, K., I.P. Michael, P. Mohseni, R. Desai, M. Mileikovsky, R. Hamalainen, R. Cowling, W. Wang, P. Liu, M. Gertsenstein, K. Kaji, H.K. Sung, and A. Nagy, piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature, 2009. 458(7239): p. 766-70.
8. Kaji, K., K. Norrby, A. Paca, M. Mileikovsky, P. Mohseni, and K. Woltjen, Virus-free induction of pluripotency and subsequent excision of reprogramming factors. Nature, 2009. 458(7239): p. 771-5.
9. Stanbridge, L.J., V. Dussupt, and N.J. Maitland, Baculoviruses as Vectors for Gene Therapy against Human Prostate Cancer. J Biomed Biotechnol, 2003. 2003(2): p. 79-91.
10. Tani, H., M. Nishijima, H. Ushijima, T. Miyamura, and Y. Matsuura, Characterization of cell-surface determinants important for baculovirus infection. Virology, 2001. 279(1): p. 343-53.
11. Babendure, J.R., J.L. Babendure, J.H. Ding, and R.Y. Tsien, Control of mammalian translation by mRNA structure near caps. Rna, 2006. 12(5): p. 851-61.
12. Mahonen, A.J., K.J. Airenne, S. Purola, E. Peltomaa, M.U. Kaikkonen, M.S. Riekkinen, T. Heikura, K. Kinnunen, M.M. Roschier, T. Wirth, and S. Yla-Herttuala, Post-transcriptional regulatory element boosts baculovirus-mediated gene expression in vertebrate cells. J Biotechnol, 2007. 131(1): p. 1-8.
13. Yu, J., K. Hu, K. Smuga-Otto, S. Tian, R. Stewart, Slukvin, II, and J.A. Thomson, Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences. Science, 2009.
14. Shan, L., L. Wang, J. Yin, P. Zhong, and J. Zhong, An OriP/EBNA-1-based baculovirus vector with prolonged and enhanced transgene expression. J Gene Med, 2006. 8(12): p. 1400-6.

Tags: bacmam, baculovirus, BVES, iPS cells, iPSC, iPSCs, non-integration, stem cells, therapeutic iPS cells

Protocols for Using Human Fibroblasts Expressing Human bFGF as Feeder Cells for iPSCs

New Product of the Week: Anti-GFP Polyclonal Antibody 100ug ABP-PAB-PAGFP10 $175.00.

Allele Biotech has introduced the highly efficient GFP-Trap for GFP fusion protein pull-down, and a monoclonal anti-GFP antibody for detecting GFP-fusion proteins after Immunoprecipitation with GFP-Trap. Just launched this week, the anti-GFP polyclonal antibodies provide an alternative method for analyzing the isolated proteins.

Pre-announcement: Allele Biotech will launch a FAQ and a User Forum online where you can also find common protocols in focus areas and exchange ideas with us or others.

1. Thaw one vial of irradiated feeder cells by swirling gently in 37oC water bath until all of the contents are thawed. One vial of 2×10^6 cells is sufficient to prepare two10-cm dishes, or two 6-well or 12-well plates (about 3-4×10^4/cm2).
2. Spray vial with 70% ethanol and wipe dry before placing in tissue culture hood.
3. Gently add 1 ml prewarmed feeder cell medium (alphaMEM or DMEM/F12 with 10% FBS), mix with contents of cryovial and transfer into 15-ml conical tube containing 4 ml prewarmed feeder cell medium.
4. Centrifuge the cells at 200g at room temperature for 5 min and discard the supernatant.
5. Resuspend the feeder cells in 12 ml feeder cell medium. If using a 6-well plate: add 1 ml of feeder cell suspension to each well of the 6-well plate containing 1 ml fresh feeder cell media per well. If using a 10-cm tissue culture dish: add 6 ml of feeder cell suspension to 10-cm tissue culture dish containing 6 ml fresh feeder cell media. If using a 12-well plate: add 0.5 ml feeder cell suspension to each well of 12-well plate containing 1 ml fresh feeder cell media per well. Gently shake the dish left/right and up/down 10-20 times without swirling the plate to evenly distribute the cells across the plate.
6. Incubate the cells in 37 1C, 5% CO2, overnight.
CRITICAL STEP When moving the feeder cell plates from the tissue culture hood to incubator, do not swirl the medium, as this tends to cause the cells to accumulate in the center. Immediately after placing the plates in the incubator, slide the plates forward and backward (2–3 cm) two times, then left to right (2–3 cm) two times to ensure equal distribution of the cells. Use within 5–7 days.
7. Split stem cells (~2.5 x 10^5 to 5 x 10^5 cells, or ~10% confluence) into plate with feeder cells: aspirate medium from ESC or iPSC, wash with PBS and add 0.5 ml of 0.05% trypsin. Incubate at 37oC, 5% CO2, for 5 min.
8. Inactivate trypsin with 3 ml stem cell medium (e.g. DMEM + 20% knockout serum replacement), and collect cell clumps in 15-ml conical tube avoiding making single cell suspension because ESC tends to die in single cell form.
9. Centrifuge at 200g at room temperature for 4 min.
10. Aspirate feeder medium from feeder plates (cells incubated in Step 6), rinse with one ml of stem cell medium and add 5 ml of stem cell medium and return to incubator.
11. Aspirate and discard supernatant from the conical tube in Step 8, resuspend cells in 5 ml stem cell medium, gently dispense the cell pellet three times, add to feeder cell wells or dishes.
12. Incubate stem cells grown on feeder cells at 37oC, 5% CO2, for 48 h.
13. Aspirate medium and replace with stem cell medium every day; if iPSC colony number is low, replace medium every two days.

Tags: bFGF, DMEM/F12, FAQ, feeder cells, forum, human fibroblasts, iPS cells, iPS protocol, iPSCs, New Product of the week, protocol, stem cells

FAQ About Feeder Cells for Stem Cells –Part One

The cost of preparing feeder cells for induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs) is mainly due to 1. serum and media, 2. labor for growing and treating cells, and 3. expenses for freezing media and vials. Ready-to-use feeder cells saves one important labor-intensive step of iPSC generation, it should be an important help for iPSC and stem cell researchers. We know that most of our colleagues are tired of preparing fresh early passages of MEFs and treating them with expensive mitomycin C or finding an irradiator to pre-treat the MEFs. A lot of iPSC researchers lost iPS stem cells due to the lack of patience in handling MEF feeders. The offering of Allele’s feeder cell product line is really an easy solution and convenience to iPSC researchers.

Question 1: There are companies offering drug-resistant feeder cells such as MEF cells expressing neo-, puro-, or hygromycin-resistance genes. Is it important to have such drug-resistance genes when choosing feeder cells?

Adding drug resistant markers to these cells should not be necessary because iPSCs grown on feeder cells are usually not cultured in antibiotics-containing medium. The feeder cells will not be selected by drug resistance nor will they contaminate iPS cells since they can not propagate after irradiation. However, for those who do need to use drug selection for any reason, we will provide drug-resistant feeder cells upon request.

Question 2: There are publications showing the use of cells lines as feeder cells instead of primary fibroblasts, e.g. SL10, MRC-5, STO. Are there any advantages of using these cell lines?

Not really. Handling primary cells requires certain amount of experience and may be tedious; using cell lines, on the other hand, would be easier for preparing feeder cells. We provide feeder cells from immortalized early passage human foreskin fibroblasts at prices often lower than those from cell lines.

Question 3: Should I choose fluorescent protein expressing feeder cells for easy separation from iPSCs?

You do not need to include fluorescent protein in feeder cells, as feeder cells are quite different in morphology from iPS cells or ES cells. In fact, many labs use iPS factors that are co-expressed with fluorescent markers, in which cases feeder cell expressed fluorescent proteins will confuse the readout.

Question 4: What are the main advantages of using bFGF-expressing feeder cells?

Our bFGF-feeder cells not only eliminate the needs for added recombinant bFGF to stem cell cultures, but also form very nice cell lawn to serve iPSC colony formation because of their strictly controlled passage and growth conditions. We have used these cells without coating dishes with gelatin and obtained nice iPSC colonies.

Preview: Next Part of FAQ on Feeder Cells: choosing mouse or human fibroblasts, selecting iPSC colonies…

Announcement: An audience-orientated User Forum will be added to Allele Biotech webpages so that people can freely discuss or review products and technologies. A distilled version of discussions will be presented in a related but separate FAQ section, which will also include all Allele eNewsletters sent to our contacts about every quarter. Look for the links on www.allelebiotech.com in coming weeks.

Tags: bFGF, biology FAQ, biology forum, biotech enewsletter, ES cells, ESC, ESCs, feeder cells, human fibroblasts, iPS cells, iPSC, iPSCs, irradiation, MEF, MEFs, mitomycin C, stem cell culture

Nobel Prize in Medicine Awarded to Discovery of Telomere and Telomerase

Elizabeth H. Blackburn, Carol W. Greider and Jack W. Szostak are credited with discovering how telomeres work and the function of telomerase. “As cells divide, chromosomes need to be replicated perfectly. Work by the researchers determined that telomeres protect DNA from degradation in the process, and that telomerase maintains the telomeres,” as reported by CNN.

Carol Greider was a student of Blackburn, and Szostak collaborated with the Blackburn group 20 years ago and has since left that field. Still remember going to Blackburn’s seminar as part of the molecular biology seminar series at USC in the early 90’s, and reading Szostak’s papers on aptamer selection while designing RNA aptamer selection schemes (SELEX) to find substrates of pre-mRNA splicing factors.

By JW

Product related note: Human telomerase gene TERT is provided on lentiviral vectors to increase efficiency of generating iPS cells.

Tags: aptamer, cancer research, chromosome, iPS cells, iPSC generation, lentiviral vector iPSCs, Medicine, Nobel Prize, Selex, telomerase, telomere