Cell Cycle Assays-Part I

This is the first part of a series of blogs about using fluorescent proteins in cell based assays with established examples, a common theme here at the AlleleBlog.

FUCCI Cell Cycle Sensor

The FUCCI Cell Cycle Sensor is composed of a red (RFP) and a green (GFP) fluorescent protein fused to different regulators of the cell cycle: cdt1 and geminin.

During the cell cycle, these two proteins are ubiquitinated at different time points by specific ubiquitin E3 ligases, which tag them for degradation in the proteasome. The E3 ligases’ activities are regulated temporally and result in the biphasic cycling of GERMINI and CDT1 levels during the cell cycle. In the G1 phase of the cell cycle, GERMINI is degraded; therefore, only CDT1 tagged with RFP is present and appears as red fluorescence within the nuclei. In the S, G2, and M phases, CDT1 is degraded; only GERMINI tagged with GFP is present, resulting in cells with green fluorescent nuclei.

During the G1/S transition, when CDT1 levels are decreasing and GERMINI levels increasing, both proteins are present, so are the tagged fluorescent proteins. When the green and red images are overlaid, nuclei fluoresce yellow. This dynamic color change, from red-to-yellow-to-green, represents the entire cell cycle. This representation can be used to study the effects of elements that may influence cell cycles.

Sakaue-Sawano A, Kurokawa H, Morimura T, Hanyu A, Hama H, Osawa H, Kashiwagi S, Fukami K, Miyata T, Miyoshi H, Imamura T, Ogawa M, Masai H, Miyawaki A.Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell. 2008 Feb 8;132(3):487-98.

CCNB1-CyclinB(NT)-GFP

In late S phage, CCNB1 promoter will be switched on to drive the expression of Cyclin B N-terminus-GFP expression; thereafter the fluorescent signal will be switched off at the destruction box in Cyclin B N-terminus at the end of Mitosis phase. During the intervening phase the fusion reporter protein will translocate from cytoplasm to nucleus by the cytoplasmic retention signal in the Cyclin B N-terminus.

Thomas N. Lighting the circle of life: fluorescent sensors for covert surveillance of the cell cycle. Cell Cycle. 2003 Nov-Dec;2(6):545-9.

GFP-PCNA/YFP-PCNA
GFP-PCNA, a fusion of GFP and PCNA, has been widely used as a convenient tool to monitor the progress of S phase. At the onset of S phase, GFP-PCNA translocates into the nucleus; at mitosis the nuclear envelope breaks down and the nuclear accumulation of PCNA-GFP dissipates.

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Tags: cell based assays, cell cycle, cell cycle arrest, DNA-repair, Fluorescent proteins, M phase, mitosis, S Phase

Purifying DNA without Membrane Binding

Purifying DNA from natural samples or biochemical reactions is one the most frequently performed experiments in virtually all molecular biology labs. Binding DNA to silica membranes in chaotropic binding buffers is the currently prevailing method, pioneered by Qiagen. Before Qiagen columns and the similar columns from a number of companies, including Allele, there was the silica resin, mostly from Promega. Before silica, it was phenol extraction or CsCl gradient.

Silica-based technology has been around for more than a decade, and it is time for a new generation of technologies that are more convenient and efficient than silica membrane to take center stage of DNA purification, especially given the fast-paced advances in polynucleotide analysis in microarrays and deep sequencing. Solid Surface Reversible Binding (SSRB) technology should be a shining star in coming years. The process is simple: DNA or RNA molecules in a simple binding buffer bind to the surface of plastics of any size and shape (PCR tube, 2.0ml eppendorf, 96-well plates, even 15 ml or 50 ml conical tubes) that is treated by a special process, washed, and eluted in any volume of water or even downstream reaction buffers. The utmost convenience is that the downstream reaction can be performed in the same tube!

This process is different from the electroreversion type of binding and releasing that requires buffers of different and extreme pH. Allele Biotech has started marketing SurfaceBind PCR purification kits, and will roll out products that are specifically tailored for genomic DNA, mRNA, size-differentiated DNA or RNA, DNA or RNA from fixed samples, from different species, etc. The convenience and cost-efficiency of these systems will provide significant contributions to the broad scientific community.

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Tags: deep sequencing, DNA purification, genomic DNA, microarrey DNA sample, miniprep, miRNA, nextgen sequencing, PCR cleanup, PFA linked, RNA purification, silica membrane, surface resersible binding

DNA Repair Pathway Factors in Cell-Based Screening for Restoring Patients’ Sensitivity to Cancer Therapies

Cancers undergoing therapies may develop resistance to treatment. Many current cancer treatments, such as cisplatin, function by creating DNA damage, particularly to fast-dividing cells, i.e., most cancer cells. These treatments may be rendered ineffective by DNA-damage response pathways. Cancer resistance to therapies may come from increased activity in nonhomologous end joining, decreased functions of mismatch repair, or reactivation of the Fanconi anemia (FA)/BRCA DNA-damage response pathway, etc. Ironically the loss of function of some of these DNA-damage repair factors may have partially caused the cancer formation in the first place. Regaining their functions in cancer cells possibly contribute to drug resistance. Molecules that disrupt FA/BRCA pathway or other DNA-damage responses could be used to help restore therapy sensitivity.

Like many proteins that function in DNA-damage repair complexes, FANCD2, a member of the FA pathway factor group, is targeted towards chromatin following damage to DNA in a process called foci formation. There have been recent studies that monitored the foci formation of GFP-FANCD2 in small molecule library screening and identified inhibitors to FANCD2 as candidates for a cancer therapy sensitizer. The assays can be improved in a number of ways. There are fluorescent proteins (FPs) that are much brighter than EGFP for increased sensitivity. For instance, the monomeric green FP mWasabi is about 2-3 fold brighter than EGFP, with narrower emission peak, and is more stable under acidic environment. The newly developed lancelet YFP (LanYFP, developed/introduced by Allele Biotech) is astonishingly 10 times brighter than EGFP. Since it has a longer excitation and emission wavelength, it should inherently have a better signal to noise/background ratio compared to EGFP because cells autofluoresce less in long wavelengths. The improved brightness would also help in this respect. The fold difference between foci and LanYFP background will be the same as EGFP, but the contrast will still probably be better because of less autofluorescent background and significantly higher fluorescence reading in foci.

Other factors that may be used as a screening target when fused to effective FPs may probably include:

1) Homologous recombination (HR)
a. End Resection
MRN complex (MRE11, RAD50, NBS1)
CtIP, RPA, ATM, ATR, Exo1, BLM, RMI1, TopIIIa, DNA2, BRCA1
b. Synapsis
RAD51, BRCA2, PALB2, RAD51B, RAD51C, RAD51D, RAD51AP1, XRCC2, XRCC3, RAD54, RAD54B
c. DNA synthesis
DNA polymerase delta, PCNA

2) Nonhomologous End Joining (NHEJ)
Ku70/Ku80, DNA-PK, Ligase IV, XRCC4, XLF

3) Fanconi Anemia Pathway
FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, FAAP100, FANCM, MHF, FAAP24, FANCD2, FANCI, FAN1, FANCN, FANCJ, FANCM

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Tags: cancer therapy, cancer treatment, DNA-damage, DNA-repair, drug resistance, drug screening, fluorescent protein, NIH grant, RNAi

Expression of iPS Factors from Transfected mRNA

Differentiated cells can be reprogrammed to pluripotency by enforced expression of certain combinations of stem cell-specific protein factors in them. The power of this method was first demonstrated by Yamanaka’s group using retroviruses carrying Oct3/4, Sox2, c-Myc, and Klf4. Alternative factors such as Lin28 and Nanog, and additional factors such as the human telomerase gene hTert and shRNA against p53 were also shown to contribute to reprogramming. From the very beginning it was realized that viral integration would pose a major problem in using the induced pluripotent stem cells (iPSCs) for clinical purposes. There have been multiple attempts to circumvent this problem by using non-integrating vectors such as plasmid, minicircle DNA, adenovirus, baculovirus, removable transposons, episomal DNA, or by introducing recombinant proteins with a transmembrane domain into target cells. From reports in the field and customer feedbacks it seems that retroviral or lentiviral systems are still the most efficient in reprogramming. mRNA is about the only option left unreported, until an article by Warren et al was published in Cell Stem Cell online recently.

From that report, it is clear that the reason that it took so long for RNA-induced iPSCs (RiPSCs) to appear in the literature was because synthetic mRNAs activate interferon responses in mammalian cells, reminding us of the early days of RNAi. The authors took a number of steps to reduce interferon responses, including adding a 5’-cap (actually a fairly standard step in in vitro transcription), using a phosphatase to remove 5’ triphosphates on uncapped mRNAs, and using modified C and U bases (5-methucytidine or 5mC and pseudouridine or psi) during T7 promoter-driven in vitro transcription. The prepared mRNA was then administered everyday for 17 days at an amount not clearly defined in the paper. The main benefit of this method is of course that there is no gene integration to alter the chromosome. The efficiency of the new method was also compared to using viral vectors and it was shown that 1.4% conversion efficiency was achieved vs retroviral systems’ 0.01% (although we have experienced better results using lentivirus, at least the 4-in-1 version).

The DNA templates used for in vitro transcription of the iPS factors were created by multiple PCR reactions and bridged ligation; it could also be done by other cloning strategies. For those excited about trying this new way of making iPSCs, the major hassle would be preparing modified mRNAs good and abundant enough for 17 consecutive transfections. Allele Biotech would like to provide custom services, before offering shelf products, for creating such mRNAs as the method sounds potentially very helpful to many researchers in the iPSC field.

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Tags: c-myc, in vitro transcription, integration-free, iPSC, iPSCs, Klf, lentivirus, modified synthetic mRNA, mRNA iPSCs, mRNA transfection, Oct, retrovirus, RiPSC, RiPSCs, Sox

Congress may let SBIR authority lapse this week

SBIR/STTR/CPP EXPIRATION LOOMS (circulated by Rick Shindell, reposted by AlleleBlog)

The SBIR/STTR/CPP now appears likely to expire on Thursday night, September 30.
Some will deny it but here’s what’s happening.

Allegedly the Senate and House were close to a compromise complete with an 8 year
reauthorization of SBIR/STTR/CPP but each time it goes back to the House (Nydia &
Day), they change the VC language to masquerade 100% VC involvement as a compromise.

Because time is so short, the Senate passed a bill (S.3839) to simply extend
SBIR/STTR/CPP through January 31, 2010. The House was going to pass it on Wednesday
with the President signing Thursday. However, the word on the street is that Nydia
Velazquez, chair of the House Small Business Committee, and her illustrious second,
Michael Day, are rejecting the bill and are poised to let SBIR expire if necessary,
at least in the short term.

It seems that Velazquez’s hope is to move the SBIR reauthorization into the lame
duck session and incorporate all her Wall Street investors’ 100% non-compromise VC
ownership and jumbo award support into a must pass, end of the year omnibus bill
that can’t be touched by her detractors.

This sounds like a script for TV, but several years ago we had a similar year end
omnibus situation involving Nydia (as ranking member) and Sam Graves (subcommittee
chair) and BIO/NVCA, but the main difference was that the small business committee
chair was Donald Manzullo who nipped it in the bud. In our scenario today we have
to look to the House leadership to do it, but it will take your involvement.

Many senior people in the democratic party called for the House to support the
Senate compromise bill H.R. 2965, but Nydia ignored those calls, as did Jason
Altmire, the creator of this infamous Altmire Quagmire. Now Nydia’s really “miffed”
because last week she tried to “scrub” H.R.5297, the Small Business Jobs Act of
2010, but the Obama administration and Speaker Pelosi rolled her over and passed it.

CALL TO ACTION

If SBIR is important to you and your company, it’s time to get serious and realize
that this program can, and will go away unless you make a big noise to let your
politician’s know how you feel. All of us are sick of this, and we’re now facing a
lapse. Eight times this program has been deemed important enough to keep going (via
a CR) but will Nydia be successful in blocking this ninth attempt?

Voting will occur in the House on Wednesday and this may be the last time until
after the election that the SBIR extension bill could voted on. That means we must
act on Tuesday, September 28.

Here are some suggestions and rationale behind them.

CALL CALL CALL the House Tuesday September 21! Call Nancy Pelosi’s office at (202)
225-4965, Steny Hoyer (majority leader) at (202) 225-4131, Nydia Velazquez (202)
225-2361, also the House Small Business Committee line (202) 225-4038

Those of you who are good democrats, call the remaining House Democratic caucus
leaders: John Larson 202- 225-2265, Xavier Becerra 202-225-6235, Jim Clyburn
(202)225-3315

Those of you who are good republicans, call John Boehner (202) 225-6205, Eric Cantor
202-225-2815

Tell them in your own words that SBIR is about to expire and is being held hostage
by Nydia Velazquez. Let them know how important continuation of SBIR is to your
business and the country. Ask them to please support S.3839 (additional temporary
extension of programs under the Small Business Act and the Small Business Investment
Act of 1958) to keep the program from lapsing this week.

I realize that I’m asking you to do something that requires a good chunk of your
time. However, at the risk of losing you as a reader I must tell you that I donate
a large share of my time to try and keep you informed about this program, and I’m
not asking you to do anything for me, only for you and others like you. We do have
some good representatives from both parties BUT they need to hear from you and
quickly.

If you’re bold ask, “I would like to know how a party can let itself get hijacked by
a few people (like Nydia) on a vitally important, highly regarded and accepted
program. This action is to the detriment of your constituents, the country, and
yes, even your own party!”

Here’s what’s going on in the back rooms (formerly smoke filled) The Senate agreed
on a 4 month extension for SBIR because they (Senate) largely (including many on the
Republican side) did not feel a reasonable bill could be passed in the lame duck
session. The Senate has offered up some huge compromises that some believe even
James Greenwood from BIO could live with. The very long shot is that with enough
pressure we might get a compromise bill passed by Thursday.

WHAT HAPPENS IF SBIR LAPSES, EVEN FOR A SHORT TIME

This is an interesting question. Theoretically those projects (grants and
contracts) that are already in place should be okay, but some not. All new unsigned
agreements would stop. Agency comptrollers may start adjusting their budgets to put
the overall 2.8% SBIR/STTR back into their own research pools. Administrative
funding for SBIR could be severely cut back. Remember, all of your grants and
contracts are “subject to the availability of funding.”

On the other hand, SBIR can be voluntary, so some agencies may choose to keep their
SBIR doors open, hoping for, or expecting the reinstatement of the program.

In any event, this is bad for you and the agencies.

The Insider will be on the Hill Wednesday and Thursday, so we’ll do a follow up
report to you asap.

Rick Shindell
SBIR Gateway
Zyn Systems
40 Alderwood Dr.
Sequim, WA 98382
360-681-4123
rick@zyn.com
www.zyn.com/sbir

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Tags: BIO, BIOCOM, congress, innovation, NIH funding, NIH grants, San Diego biotech, SBIR, SDEE, VC companies