Archive for October, 2009
Packaging lentiviruses or retroviruses is not a routine procedure that every biology lab performs even if there is need to use it. A viral packaging protocol normally begins with preparation of purified transfer plasmid DNA, a miniprep should be enough for a few transfections. The virus backbone plasmid is either co-transfected into commonly used cells with helper plasmids that provide the essential proteins required for particle packaging, or transfected into established helper cell lines that express the required proteins from integrated transgenes. After incubation of packaging cells for a couple of days, viruses are collected and tittered. Titer determination is somewhat tricky for the inexperienced. Using a control virus expressing a fluorescent protein can make this step convenient.
Commonly known facts:
1) Lentiviruses are packaged at a titer of 10^6 IU/ml without concentrating steps.
This needs update since with more advanced technologies lentiviruses can be packaged routinely at 10^8 IU/ml. With further concentrating, the titer can be easily above 10^11 IU/ml. Retroviruses can be packaged to similar titers as well.
2) Using packaging cell lines gives the highest possible titer
While packaging cell lines (such as Allele’s popular Phoenix Eco and Ampho cells for retrovirus packaging) provides maybe the most convenient method for packaging, the yield will not reach the highest potential. Packaging cell lines may also lose their capability for packaging after continued culturing, requiring periodic selection with antibiotics and functional tests, as we do here at Allele.
3) Retroviruses are always collected in one shot after transfection into packaging cells
If the transfer vector has oriP/EBNA1 episomal maintenance system, such as some of the Phoenix vectors Allele offers, the plasmids may continue to express for up to 30 days. With puromycin selection, the titer of retrovirus produced from Eco or Ampho cells can reach 10^7 IU/ml.
This week’s promotion (102509-103109): 10% off across the board of Allele Biotech’s custom services, for an example, check out our world-leading baculovirus protein expression.
New Product/Service of the Week: Introduction of Custom Viral Packaging Service. Routine titer of 10^8 IU/ml, as high as 10^10 IU/ml, option to include cloning. Signature service ABP-CS-MERV002 provides more than 200 million particles at $7/million particles. These are game-changing prices for the viral packaging service market based on superior technologies!
Allele’s New Products of the Week, Oct 19-26, 2009: DNA oligonucleotide synthesis reagents dA, dT, dC, dG controlled pore glass (CPG) beads for oligo synthesis. With previously launched CPG beads and phosphoramidites for modified oligos, this product line now provides the most essential materials for oligo synthesis by university core facilities, company internal oligo production groups, or commercial oligo providers at significantly reduced prices.
Allele’s Weekly Promotion Oct 19-26, 2009: In accordance with the launch of the above new products, all 3’ amino, thiol, Dabcyl, FAM, biotin modified oligos of 50 to 200 nmol scale are offered at unprecedented $10/modification.
What do you have available that can be added to the 3’ end of a primer/probe to stop PCR amplification?
There are a few commonly used modifications on the 3′ of an oligo to block polymerase extension, e.g. C3 spacer, amino-modified C6, inverted dT, phosphate. Although no 3′ blocking modifications are 100% effective, the amino-modified C6 offers the best result, leaving1% or less unblocked; phosphate is not as effective of a block, with up to 2% unblocked. We recommend 3’ amino group also because it is less expensive compared to other 3’ modifications if ordered from Allele Biotech.
Can you provide 5’ digoxigenin as a standard modification on your oligos?
5’ Dig is typically added by conjugating the digoxigenin group to a 5’ amino added during oligo synthesis. 5’ amino modification can be ordered from almost all oligo suppliers including Allele. You may need to add digoxigenin using a commercial kit by yourself. If you are interested in having Allele Oligo Service perform the chemical linking, email email@example.com.
Is Dabsyl a misspelling of Dabcyl?
DABCYL acid is the abbreviation of 4-(dimethylaminoazo)benzene-4-carboxylic acid. Sometimes DABSYL (4-dimethylaminoazobenzene-4”-sulfonyl chloride) is mistaken for ‘DABCYL’. They do share similar properties as fluorescence quenching agents, with minor difference in maximum absorbance, but can in general be used interchangeably in pair with fluorescent dyes such as FAM. Allele uses Dabcyl as its standard 3’ modification and, by using its own oligo synthesis reagents for adding this group, offers a price less than half of most other oligo manufacturers (check back for pricing updates next week for even lower prices). DABCYL is one of the most popular acceptors for developing FRET-based nucleic acid probes and protease substrates.
The hottest subject in the biological research equipment field has to be whole genome sequencing; hence it is no surprise that companies execute mergers and acquisitions in order to position themselves to go after their competitors in an attempt to corner this valuable market.
A bit of the background history: Illumina was started a decade ago to build DNA chip arrays by people with experience at Affymetrix, when the latter was the first and absolute leader in the DNA chip field. For years, rather than providing DNA chips, Illumina was known for generating revenue by selling oligonucleotides at 20% of the prevailing market price, essentially starting the low end oligo market. Just three or four years ago, it was a front page promotion on Invitrogen’s website to sell Illumina’s oligos through a production/shipping alliance, a cooperation previously unheard of in our field for such low price, non-commodity products. This move quite probably contributed to the decisions made by the more dedicated oligo company, IDT, to acquire local oligo production houses and move to the West coast (Allele opted out of such an acquisition and later did one of its own by taking over Orbigen and since moved into the viral systems and antibody fields). At that point when whole genome sequencing technologies were becoming mature and marketable, Illumina had performed brilliantly in out competing the previously dominant chip supplier Affymetrix, acquired Solexa, and quickly moved into the whole genome sequencing with Genome Analyzer and Genome Analyzer II, a move Affi’s management probably regretted not making.
In the years roughly around 2005-2007, Applied Biosystems, Inc. (ABI) was developing its own genome analysis equipment, the SOLiD system. It surely had a solid base to build on from its strong leadership in providing sequencer and analyzers for many years. Earlier in the year Invitrogen and ABI merged to form Life Technologies, pitching Invitrogen (now LifeTech) and Illumina in a collision course in battle for dominance in genomic analysis. In September, LifeTech brought suit against Illumina for patent infringement; in October Illumina countered with suits of its own. While the fight in court may be long and only sprinkled with occasional fireworks, the competition in the market could be fierce and should ultimately decide on whose technology is superior and offered at better prices. From the technical presentation made by sales teams to us during on site seminars, Solexa’s science sounded better. I was sitting next to Jay Flatley, CEO of Illumina at a San Diego biotech CEO dinner, and heard him predicting that the technology would advance and in a few years, one could get their own genome sequenced for about a thousand dollars, ~10% of the current cost! That’s simply innovation and competition at work. But watch out, a new wave of sequencing technologies based on single molecule capture might make the Illumina and LifeTech courtroom argument a moot point in the market.
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.
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.
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