Allele Mail Bag
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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New Product of the Week 082310-082910:
pInman-iPS Bac2Mam viruses, email iPS@allelebiotech.com for details.
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Promotion of the Week 080910-081510:
Thomson set of iPS vectors on lentivirus, send in order this week get 20% discount. Email iPS@allelebiotech.com for details, with promotion code V082910.
Allele’s pallet of the super star fluorescent proteins
“Photoblog”–just some fun pictures from our notebooks.
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The brightest cyan, green fluorescent proteins, and the brightest ever FP in LanYFP!
Ain't they pretty?
These fluorescent proteins are representatives of the growing family or high quality, new generation FPs engineered to enable experiment previously deemed impossible.
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Cells infected with lentivirus carrying mWasabi. Lentivirus carrying LanYFP will make most cells much more brighter than this.
The mWasabi is stimulating
The brightest green fluorescent protein with excellent photostability, carried on 10e8 TU/ml high titer lentivirus.
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The LanFPs express well in bacteria.
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.
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The FPs fold so strongly that they fluorescence even in SDS-PAGE.
Can you see the FP bands in the SDS PAGE?
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FPs in SDS PAGE–a closer look
Can you see them now?
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FPs in gel cassette over UV lights
Invincible FPs
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FPs in gel cassette under blue LED
Fluorescence in SDS page under blue LED
The purified FPs can be used as “real time” protein markers.
New Product of the Week 07/26/10-08/01/10: pCHAC-mWasabi-C for expressing mWasabi fusion through retroviral vectors.
Promotion of the Week 07/26/10-08/01/10: Get 3′ TAMRA & BHQ oligo mods for $45 ea & 3′ Dabcyl mod for $20 50 nmol syn scale only/while supplies last- use dbtkrm0726
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).
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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.
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New product of the week:
iPS specific gene promoter-fluorescent protein reporter lentiviruses.
GPCR: problems and resolutions in hihg content screening
A review in the December, 2009 issue of Nature Methods, “GPCR: insane in the membrane” by Michael Eisenstein impressed me greatly. It presents a clear and wide view of the GPCR research fields, from the basic background knowledge, to difficulties and current solutions in real cases, to future development. After careful reading and thinking, I felt that there were still several questions left to for me to pounder.
1) The Plot Thickens
“In many cases, you identify a GPCR as a target based on physiological data, and the receptor might be expressed in the brain in a particular neuron, but then you perform a high-throughput screen in over expression, immortalized cell lines that are nothing like the cell in which the receptor normally resides,”This is the inherent problem for all the engineered and artificial cell based assays most widely used in GPCR research right now. As stated in this review, “There are so many ways to be misled by using an over-expressed receptor in a non-native cell line”, it was shocking to me, making me realize that cell line screening does not only have a problem of getting raw data of low quality, but also a problem of being seriously misleading, a problem of direction rather than efficiency.
Directed differentiation of iPS cells and ESCs might be one of the potential solutions to this problem. However, from our experience in iPS cells dedifferentiation and differentiation at Allele Biotech, as a main vendor of iPS cell reagents, it is hard to make this platform in current situation suitable for high content analysis of GPCR. As much as we wish and believe, like many other researchers and the general society, that stem cells will contribute significantly also to the drug screening field, much work needs to be done before that happens.
Primary cells could be a good alternative. Based on data from Allele Biotech’s trade partners in the primary cell business, the primary cells market has been expanding dramatically since late last year. I believe that the utility and advantage of primary cells have been increasingly appreciated. However, there are still some problems with using primary cells in GPCR studies. For example, neurons are hard to obtain and can not amplify in vitro, in which cases there seems to be no chance to satisfy the need by screening. In fact, neuron research is one of the main GPCR research fields. Therefore, this is a must-fix problem.
How about “Immortalized Primary Cells”? The immortalized primary cells maintain the properties of primary cells well, relative to established cell lines. We have built our own advanced immortalization technology, based on which new products and services will be released in the first season of next year. During the last several years, we have successfully immortalized immunological cells and cancer cells. WE have yet to have the experience of working with neurons. Anyone who has interests of cell immortalization is welcome to contact us for collaboration or custom service. “Introducing Cost Effectiveness to your research”, just as the slogan of Allele Biotech, we will be proud to serve you with cutting-edge technology and cost effectiveness!
2) Without a mark to be continued…
By Niels Yuhui Ni, researcher at Allelle Biotech
mTFP1 is an excellent FRET donor
Because of its excitation and emission wavelength, sharp excitation and emission peaks, high quantum yield, and exceptional photostability, mTFP1 has always been considered a very good Forster resonance energy transfer (FRET) donor (1). More recently, several groups have investigated the use of mTFP1 in various FRET experiments and imaging modalities and have shown that mTFP1 is indeed one of the best choices (2, 3, 4).
In one recent publication, Padilla-Parra et al (2) tested a number of different FRET couples to determine which was the best for fluorescence lifetime imaging (FLIM)-FRET experiments, and found that the mTFP1-EYFP pair was by far the best pair for FLIM-FRET. This group also confirmed that the fluorescence lifetime decay of mTFP1 fits well to a single exponential, and that the time constant for this decay is unaffected by photobleaching, making mTFP1 an excellent choice for any kind of fluorescence lifetime imaging applications, including FLIM-FRET. This group also notes that it is likely that the use of Venus or mCitrine variants in place of EYFP would improve the performance of this FRET pair even further.
In a mathematical analysis of the potential FRET efficiency of mTFP1 with Venus YFP, Day et al. (3) showed that compared with Cerulean (currently the brightest cyan Aequorea GFP variant), one can expect up to 17% better FRET efficiency using mTFP1. This group went on to characterize the mTFP1-Venus pair in live-cell FRET and FLIM-FRET experiments and showed that it worked as predicted in both cases. They also note that mTFP1 has superior brightness and photostability when compared to Cerulean in live cells, which is consistent with all in vitro data reported previously (1). In a related paper, Sun et al. (4) demonstrated that mTFP1 is also an excellent FRET donor for the orange fluorescent protein mKO2.
Together, these recent independent studies confirm that mTFP1 among the best options when choosing a fluorescent protein as a FRET donor. With its proven track record of successful fusions, mTFP1 is also an excellent all-around performer that will enhance almost any live-cell imaging experiment.
(1) Ai et al., (2006) Biochem. J. 400:531-540.
(2) Padilla-Parra et al., (2009) Biophys J. 97(8):2368-76.
(3) Day et al., (2008) J Biomed Opt. 13(3):031203.
(4) Sun et al., (2009) J Biomed Opt. 14(5):054009.
AlleleBlog Admin, by Nathan Shaner
Video of the month (NEW!): Protein Expression Systems on youtube (http://www.youtube.com/watch?v=n81orbUebsQ) and at our protein expression page.
Discount of the week (Dec 14-20): 15% off Phoenix Retrovirus Expression System 2.0 (with selection medium provided)
New product(s) of the week: 48 fluorescent protein fusions on ready-to-infect virus that get into primary mammalian cells as subcellular markers (http://www.allelebiotech.com/shopcart/index.php?c=197&sc=34), 20 infections, only $249 for a limited introduction time.
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