Monitoring the Undifferentiated Stage of Stem Cells—the Pluripotency Markers

Human embryonic stem (ES) cells or induced pluripotent stem (iPS) cells promise to serve as an unlimited source for transplantation or tissue-specific differentiation. However, obtaining and maintaining stem cells are very difficult tasks for multiple reasons. For instance, most stem cell lines tend to spontaneously differentiate in culture, and even if the cells form stem cell-like colonies, they may be of a heterogeneous population.

To identify pluripotency of stem cells, expression of stem cell-specific marker genes (i.e. Oct-3/4, Sox2, Nanog, Rex-1) is monitored by RT-PCR. Alkaline phosphatase activity and methylation profiles of promoters of pluripotency-relevant genes are often analyzed as well. Compared to murine cells, it is noticeably more difficult to obtain human iPSCs, of which stem cell-like colonies sometimes turn out not to be pluripotent cells. We highly recommend testing iPSCs, especially human iPSCs, with antibodies against stage-specific embryonic antigens such as SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81.

However, all of these methods require cell destruction or fixation for analysis, therefore, are inconvenient and costly. Furthermore, many studies using ES or iPS cells involve differentiation of stem cells into different lineages, a method for observing live cells to know their undifferentiation/differentiation stages would be very helpful. There have been a number of publications using murine Oct-4, Nanog, and Rex-1 promoter driven fluorescent proteins as markers for pluripotency tests [1-3]. Allele Biotech provides, under its iPS product line, packaged and validated lentiviral particles that would insert these 3 promoter-FP reporters into the stem cells. Although currently these promoters are of mouse sequences, their use in human stem cells have been reported.

New product of the week 01-25-10 to 01-31-10:

All-In-One-Vector: Human OSKM Lentiviral Paticles, with Oct-4, Sox-2, Klf, and c-Myc all expressed from a single virus, ready-to-use.

• Promotion of the week:

human iPS cell detection primer set, the same as the landmark Yamanaka paper [4] on creating human iPS for the first time.

1. Da Yong WU, Zhen YAO (2005). Isolation and characterization of the murine Nanog gene promoter. Cell Research, 15 (5): 317–324.
2. Rachel Eiges, Maya Schuldiner..et.al (2001). Establishment of human embryonic stem cell?transfected clones carrying a marker for undifferentiated cell. Current Biology 11: 514–518.
3. Guangjin Pan, Jun Li, Yali Zhou, Hui Zheng, and Duanqing pei (2006). A negative feedback loop of transcription factors that control stem cell pluripotency and self?renewal. ASEB Journal 20: E1094? E1102
4. Takahashi et al, Induction of Pluripotent Stem Cell from Adult Human Fibroblasts by Defined Factors (2007). Cell 131, 861-872

Tags: differentiation, fluorescent protein, iPS, iPS lentivirus, iPSC, iPSCs, lentivirus, pluripotency reporter, promoter, promoter-FP, stem cells, undifferentiation stage

Submit Your Pictures and Enter to Win $100 in Cash

Allele Biotech is holding another fun contest to give back to our loyal supporters and everyone has the chance to win $100 in cash! We are looking for interesting and relevant pictures, illustrations, and computer generated art that pertains to any Allele Biotech Product or service to replace the flashes on our homepage. If it is chosen to be used we will give you $100 cash!

Check out our technical data sheets, read our past blogs, and even use your own lab experience and impressions with Allele Biotech Products to come up with your entry. There are over 1000 products to choose from so everyone should have lots of inspiration!

All you have to do to enter is submit your picture to our facebook inbox! The picture has to be of your own creation and/or one to which you own exclusive rights. Response time and prizes will take about a week.

Let your creative juices flow and you could get $100 in return. Your picture could be funny, serious, or even super nerdy!

Used to promote our Sapphire(TM) Baculovirus Expression Service

All photos, illustrations, and computer generated graphics (aka. “the picture”) submitted must be lawfully owned by entrants who submitted them. Pictures that are not chosen will not be used in any way by Allele Biotech. Winners may collect their prize via cash, check, or a one-time Allele credit of $100 good toward any Allele Biotech purchase. Allele Biotech has full rights to pictures selected for use in Allele advertising and winners must forfeit any future rights to the picture. Contest to run indefinitely and to be terminated at any time.

Tags: cash prize, contest, submit pics for cash, win $100

New Product for 1-18-10 to 1-24-10 High Throughput DNA Oligos!

Need a plate of high quality oligos fast and for a great price? Allele’s newest product can help…

Introducing High Throughput DNA Oligos!

Anywhere from 48 – 96 desalt oligos per plate

Oligos 20 – 50 bases long

Available in two synthesis scales:

25 nmol scale only 13 cents per base

50 nmol scale only 17 cents per base

Every oligo is strictly controlled for quality:

Oligo quality is verified using MALDI-TOF mass spectrometry or Electrospray Ionization Mass Spectrometry.

Shipped or hand delivered in 5 days!

Oligos can be provided normalized at a specific concentration with fixed or variable volumes. Concentration options depend on the oligo length, purification, and scale of synthesis being ordered.

No minimum number of plates per order required!

To order, simply email your oligo sequences and names, indicate 25 nmol or 50 nmol, concentration preference in excel or notepad format to oligo@allelebiotech.com A template will be posted online here soon, please check back.

Allele…Continuing to introduce cost efficiency to research!

Tags: DNA oligo, high throughput oligos, plate oligos, price for oligos, save on reasearch

New product of the week 01-11-10 to 01-17-10

Light-driven proton pumps were used to to silence the activity of genetically specified neurons in a report by Chow et al. in the latest issue of Nature. Genetic delivery of proton pump proteins, Arch and Mac, will be of significant help to neurology labs. We have started put these genes into Allele Biotech’s ready-to-infect lentivirus and retrovirus, which will be available for shipping within a week or two. Allele Biotech decided to list these viral particle products as this WEEK’S NEW PRODUCT among a good number of choices to reflect our model of doing business and science: move fast and stay on the front edge of multiple moving fields.

New product of the week 01-11 to 01-17-10: arch and mac Expression Lentiviral Particles Cat # ABP-RP-TLCARC or TLCMAC.

Promotion of the week: 20% off our most popular TA cloning kit with top of the line competent cells, as announced every Monday through our social networks. Follow us there is you want to get them in time to make a purchase at the deeply discounted prices.

Tags: arch, channelrhodopsins, Chow et al., Halorubrum sodomense, ion modulator, Light-driven, mac, optogenetic, proton pumps, silence of neurons, silencing of neural activity

GPCR: problems and resolutions in high content screening, Part II

2) Without a mark (continued from Part I by Niels Yuhui Ni, MD Ph.D. of Allele Biotech)

In this section, the author focused on the “Label-Free” system. “A newer alternative that has given hope” because these systems are closer to the real cellular conditions most GPCR studies are meant to address. However, label-free systems must depend on complicated detection system for high content analysis. As commented, “For some scientist though, this technology is simply still too new and for now, too expensive for many categorical assessments…” While I am a believer in label-free detection, however, here is my question, for this group of scientists, are there any good alternatives now, before the label-free systems become more accessible? Using the traditional over-expression cell lines seems less and less attractive (see Part I under the same blog topic here)? A system that combines immortalized primary cells and a non-integrated expression system could be a nice system that does not require high end equipment or heavy commitment in technology development especially if the components were commercially available. The third, critical component of this system could be the application of newer, brighter and monomeric and thus less toxic fluorescent proteins or FRET pairs as sensors. Obviously part of the reason that I thought about such a system was because our research team at Allele Biotech has the background in all 3 components, whereas others might have their own preferred methods. To me, it seems that immortalized primary cells present a renewable cell source, and for non-integration delivery, we prefer Baculo viral delivery vehicles for mammalian infection (called BacMam by some). Both platforms are being offered as products or services already or in the pipeline teed up for launching.

Baculo2Mam non-intergrated viral delivery system:
Many people may know about Baculovirus such as in the Bac-to-Bac system from Invotrogen or the Sapphire baculovirus system from Orbigen (acquired by Allele Biotech). But how many of us know that even though mammalian cells are not the nature host of baculovirus, they still can be infected with modest modifications on the virus. Both the safety and efficiency of Baculovirus for mammalian use are superb. Based on the data from our customers’ projects, most protein expression require baculoviral protein expression in insect cells, only about 10% require Baculo2Mam. We actually feel a sense of responsibility for introducing this technology to as many as researchers as possible.

The following list shows some of the advantages of Baculo2Mam I can list right now, for more details check back on our blog articles in coming days or contact us at any time for discussion.
1) Baculoviruses are Risk Group 1 or biosafety 1 agents.
They are produced in insect cells and can not replicate in mammalian cells. They express genes in human or mouse cells in non-integrated state for about 2 weeks (varies in different cells).
2) Baculoviruses can be easily generated in high titer and production rapidly scaled-up.
That is when compared with other viral systems. For example, baculovirus is a budding virus that is released into cell medium, unlike adenovirus that requires lysing cells during productions. Allele Biotech now provides Baculo2Mam viral packaging service at an affordable price for routine use. Your viral clones can be stored in Allele Biotech’s Baculo2Mam virus bank; if you need the virus again, you can just order a production service at an even lower price.
3) Broad host cell range including many primary cells.
Many terminally differentiated primary cells such as neuron, adipocytes have been tested in Allele Biotech’s lab as target for modified Boculovirus. To assess the infection efficiency, you can order a pre-made Baculo2Mam-mWasabi GFP or Baculo2Mam-LanRFP control for a test run. Once you order custom or regular Baculo2Mam products, the cost of the control will be credited back.
4) Up to now, little or no cytopathic effects were observed of using baculovirus in mammalian cell cultures.
5) Other points that may be related to GPCR assays in relevance to mimicking natural cellular environment:
a) Delivery of biosensor to cells just prior to assay without establishing cell lines
b) Large insert capacity for expressing long cDNAs.
c) Multiple virus transductions, simultaneous delivery of multiple genes
d) Expression level can be adjusted by viral titer
e) Finally, Baculo2Mam Viruses can be stably stored at 4oC for up to 3 months, and even longer as seed stocks (i.e. titer will drop but still amplifiable).

Promotion of the first week of 2010:

in the spirit of celebrating Allele’s 10th anniversary and in line with the ongoing “get oligos free for a month” program, we offer $20 off for oligos on 3’ TAMRA or FAM modifications.

New product of the week:

iPS specific gene promoter-fluorescent protein reporter lentiviruses.

Tags: bacmam, baculovirus for mammalian cells, biosensors, cell based assay, cell imortalization service, Fluorescent proteins, GPCR, GPCR assays, immortalizaed primary cells, label-free detection, membrane based assay, viral packaging service