Commonly Known Facts About Viral Packaging -That Might Not Be Correct…

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!

Biotech Companies Can Now Make Company News Releases Through AlleleNews

AlleleNews publishes the most current, succinct, and to-the-point news and views about biological research that a typical bench scientist would be interested in reading “in-between-experiments”. The full news articles are all original, prepared by senior researchers at Allele Biotech; the short links to top publication news are carefully selected by the AlleleNews staff everyday. The articles are written based on what aspects a graduate student or a postdoc would like to know about a recent paper, instead of what a news agency would typically like them to read. For instance, AlleleNews articles often describe what significance a publication has in which field, how the concept was established and experiments designed, what major distinction it has compared to previous studies or results from other groups, and what areas might be weak points. Less mentioned are the descriptions of the labs, the departments, the institutes and what the officials were quoted as saying about the significance of the discovery. Because of this distinction, the number of readers has been growing at a rapid pace judging from page visits.

AlleleNews site also serves as one of the online platforms for company news releases and user feedbacks. It will be for the benefit of the readers as well as Allele Biotech to further enhance the contents and the functions of this news platform, therefore, a new policy is hereby formally announced: AlleleNews will be open for other companies to make news releases or company announcements. While the AlleleNews administration reserves the rights to make necessary changes, this service will be free to all companies, large or small, with no limits on number of releases. To release any news related to biomedical research or business activities, please email news@allelebiotech.com.
AlleleNews Admin

Q&A About Choosing Modified Oligos

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.

Question1:

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.

Question2:

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 oligo@allelebiotech.com.

Question3:

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.

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 2×106 cells is sufficient to prepare two10-cm dishes, or two 6-well or 12-well plates (about 3-4×104/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 105 to 5 x 105 cells, or ~10% confleuce) 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, 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.

WSJ article "Deficits and the Chinese Challenge"

It was recommended by fellow members of BayHelix, an association of professionals in the Biotech/Pharma industry most with training in the US after college.

This "interesting article for BayHelix" as mentioned by one member, in my opinion is one of the rather shallow and narrow-minded articles in the WSJ, taking Wall Street Journal Asia as WSJ's Asia edition. World affairs such as wars and survival of nations go way beyond debts and lending. America did not gain power and respect by lending money-- think of the Marshall plan to rescue Germany from the Soviets. America's contributions to both world wars and the prevention of the 3rd one so far certainly included money, lots of it, and blood, lots of that too. Americans took part in so many wars around the world, and the only territories they occupied are those that were used to bury the fallen American soldiers as many of us have already known. Creativity and innovation are results of an open, fair, and competitive society, not a plan of a nation’s leaders or its people’s collective will. Can anyone find another place where competition in a society is more based on one's personal talent, effort, and hard work than in the US? There is no absolute fairness and justice, the American society as a whole is the closest thing to it.

“Fights over health care and climate change are the cultural equivalent of fiddling while Rome burns.” stated in the WSJ Asia article. How about ignoring health care and climate change is historical equivalent of burning Rome by Romes own citizens. When the earth burns, do you really care that much about who still owes you debts?

-----------------
Deficits and the Chinese Challenge
– How United States supplanted the British Empire
By ZACHARY KARABELL
Printed in The Wall Street Journal Asia, October 14, 2009
The dollar's sharp drop over the past few weeks has led to considerable
anxiety about the status of the United States as the dominant force in the
global economy. Closely related to this fear is constant worry about the
rise of China and the evermore complicated relationship between Beijing and
Washington.
Most people are now aware that China is the largest creditor to a heavily
indebted U.S. government. It holds close to a trillion dollars of U.S.
Treasurys and has invested hundreds of billions more in private enterprises
in America. Even though these facts are plainly acknowledged, policy makers
and experts continue to underestimate the full ramifications of this
relationship.
Consider what happened in 1946, when a cash-strapped Great Britain turned to
the U.S. for a loan. For 30 years or more, the British had been consumed by
the threat of a rising Germany. Two wars had been fought, millions of lives
had been lost, and the British treasury was dramatically depleted in the
process. Britain survived, but the costs were substantial.
In spite of its global empire, a powerful military, and an enviable position
at the center of world-wide commerce, in early 1946 the British government
faced a serious risk of defaulting on its financial obligations. So it did
what it had done at various points over the previous decade and turned to
its closest ally for assistance. It asked the U.S. for a loan of $5 billion
at zero-interest repayable over 50 years. As generous as those terms seem
today, such financing had been almost routine in years prior. To the
surprise and shock of the British, Washington refused.
Unable to take no for answer, Britain explained that unless it received
funds the government would be insolvent. The Americans came back with a
series of conditions. They would lend Britain $3.7 billion at 2% interest,
and the British government would have to abide by the 1944 Bretton Woods
plan, which made the dollar rather than the pound sterling the reference
point for global exchange rates and required Britain to make the pound
freely convertible. Even more significantly, Britain had to end its system
of imperial preferences, which meant no more tariffs and duties on goods to
and from colonies such as India. These were not mere financial penalties:
Taken together, they meant the end of the British Empire.
Within two years, Britain had left India and was on its way to decolonizing
throughout Asia and Africa. Unable to compete with the U.S. economically and
no longer able to reap the benefits of colonial trade, Britain's military
shrank and its commerce contracted. It quickly receded from its dominant
global position and entered several decades of economic malaise. In the
1980s, Britain finally emerged as a prosperous country, but it was a shadow
of what it had been in its heyday.
The U.S. replaced Britain as the guardian of the West. As one British
official, Evelyn Shuckburgh, remarked in the late 1940s, "it was impossible
not to be conscious that we were playing second fiddle." And that was
precisely what the U.S. desired. Having supported the British for decades
and become its banker and manufacturer during two wars, at the end of World
War II the U.S. fully intended to supplant the British Empire. The loan
request provided the pretext, but by then the balance had already shifted
and Britain could have done little to reverse the tide.
By 2030—if not sooner—China is likely to surpass the U.S. in the size of
its economy, though it will remain on a per capita basis a much poorer
society for many years after that. Trajectories can change, but the recent
implosion of the American financial system has only accelerated China's
rise.
Given the lesson of the British Empire's demise, it would be foolish to base
current policy on the assumption that China will hit a fatal speed-bump
before it is able to supplant the U.S. And while the level of current
indebtedness is manageable for the U.S.—and in fact tethers the Chinese
closely to the U.S. economy in ways that are arguably beneficial for both
countries—the fact that these economies are currently bound together does
not mean that their interests will always be in sync.
Here, too, the British analogy is sobering. For decades, the relationship
between Britain and the U.S. was mutually beneficial, though the Americans
resented being treated as junior partners. As tension festered, the British
were consumed with the more immediate threat of Germany. But in the end it
was the U.S. that delivered the knockout blow.
The Americans have not had to deal with a true economic rival since the
British more than half a century ago. America today is as unaccustomed to
global economic competition as the British were at their apex. The U.S.
often seems lumbering and ill-suited to the demands of economic rivalry.
The only way to avoid Britain's fate and meet the challenge of China is to
reinvigorate economic life. This is a multiyear endeavor that must be done
primarily through innovation, not legislation. America needs to retool its
domestic economy to build on the global success of many U.S. companies. It
must focus on inventing new products and generating new ideas, rather than
defending the rusty industries of yesterday. Fights over health care and
climate change are the cultural equivalent of fiddling while Rome burns.
China thrives because it is hungry, dynamic, scared of failure and convinced
that it should be a leading force in the world. That is why America thrived
a century ago. Today, such hunger and dynamism seem less evident in American
life than petulance that the world is not cooperating.
The U.S. is in danger of assuming that because it has been a dominant nation
on the world stage, it must continue to be so. That is a recipe for becoming
Britain.

Intracellularly Expressed Camelid Single-Domain Antibody (VHH) Counteracts Cytotoxicity Seen in Agricultural Epidemics

The advantages of camelid antibodies with only the heavy chains have been exploited for therapeutic use or as novel research tools such as immunoprecipitation trap (e.g. GFP-Trap). These so-called nanobodies are small in size (14-15kD), highly stable, and capable of binding to epitopes that traditional antibodies are normally not able to bind. Doyle et al. published a paper in JBC last week demonstrating that when expressed inside cells (intrabody), the VHH fragment can provide antitoxicity protection against deoxynivalenol (DON) or 15-acetyl0deoxynivalenal (15-AcDON), common toxins involved in agriculture infection Fusarium.



Camelid antibodies against low molecular weight 15-AcDON were isolated by immunizing llama with 15-AcDON-BSA protein conjugate as published earlier by the same group of researchers in Canada. As demonstrated previously, the small nanobody derived from camelid antibody can be highly expressed inside target cells without causing much cytotoxicity by itself. Images generated by confocal immuno-microscopy showed that VHH is evenly distributed throughout the cytosol. The antitoxin effects were specific, effective, and apparently dose-dependent.



This report, albeit using a yeast model system instead of natural targets of the relevant plant disease, opens doors to increased tests of using VHH fragments for broader applications in agriculture and more fields other than therapeutics.


Doyle et al. 09-2009

http://www.jbc.org/cgi/doi/10.1074/jbc.M109.045047

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.


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

Allele Introduces New Version of BVES with Insect Specific IRES

Internal ribosome entry site (IRES) can be used to initiate translation of a second open reading frame (ORF) of an mRNA, providing the benefits of: 1) avoiding promoter competition in a dual promoter situation; 2) having controlled ratio of expression of two proteins; 3) placing a dominant selection pressure on the entire bicistronic mRNA and hence the maintenance of the transgene when a selection marker is placed as the second ORF.

IRES elements are located mainly in RNA viruses except certain mammalian and insect mRNA molecules. Only one DNA virus has so far been found to contain an IRES, the while spot syndrome virus (WSSV) of marine shrimp. This IRES, compared to a very few other choices known to function in insect cells such as the IRES from Rhopalosiphum padi virus (RhPV), has strong translation initiation activity (~98-99% in reference to cap-dependent initiation), insect cell specificity, and encompasses only 180 base pairs.

Allele Biotech, with its acquisition of Orbigen, is a major provider of BVES products and services with more than 10 years of experience. Allele’s featured New Products of the Week* this week are WSSV IRES containing baculovirus vectors, the sIRES (for Strong IRES from Shrimp virus) series plasmids. Currently one version is pOrb-MCS-sIRES-VSVG for pseudotyping baculoviruses (within the Emerald Baculovirus for Mammalian Expression series), with pOrb-mWasabi-sIRES-VSVG as a fluorescent protein control; the other is pOrb-MCS-sIRES-MCS for cloning a custom second cDNA. New versions in the future will include IRES driven mWasabi and other commonly used selection markers.

With a current research project for the National Cancer Institute (NCI) within the National Institutes of Health (NIH) involving development of modified BVES and mammalian protein expression and purification systems, Allele Biotech expects this product line to continue its expansion at a fast pace.

* Allele Biotech announces at least one new product every Wednesday through news release at AlleleNews or Allele Blog and social networks.