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Scientist Spotlight

Dirk Geerts, Ph.D. Interview of Dirk Geerts, Ph.D.
Sophia Children’s Hospital, Erasmus University Medical Center

Conducted August 2011

You were formally with The Amsterdam University Academic Medical Center, how did you come to be Director of core service facility for RNAi screening?
We were looking for a method to more quickly screen the function of different sets of genes (e.g. kinases, transcription factors) in the pediatric tumors neuroblastoma and medulloblastoma. So far we did this with cell transfection and isolation of stable cell line clones expressing inducible shRNA. This resulted in 10-15 such lines over a 2-3 year period of time. More speed was needed. We contacted Sigma, and I tested ~20 shRNA sets from Sigma against a similar number of your competitor’s constructs. Sigma tested better, and then we ordered 3 copies of the mouse and human TRC libraries, together with the Academic Medical Centers of the Universities of Leiden and Rotterdam. Each facility has since plated well over 5,000 clones. At the AMC, we had a little 1,000th gene ceremony on May 9th. The winner got two VIP tickes to the Rotterdam ATP tennis tournament next year – a gift from Sigma.

What are some of the implications of being able to screen larger numbers of shRNAs faster and more affordably?
We can set up more complicated biological queries, cheaper, and more quickly (e.g. the golden goal in modern cancer drug research: the synthetic-lethal). To name several examples we have actually performed or set up in the last two years: 1. Interrogating a complete signal transduction pathway to characterize the oncogenic mutation, 2. Confirming the specificity of small molecule inhibitors by RNAi knockdown of the target gene, 3. Defining the second event in a synthetic-lethal combination with a screen.

In your opinion, what is the next big thing in the world of pediatric cancer research?
Genome sequencing yielding a lot more mutated genes to target, and research with tumor initiating cell cultures for more directly targeting the tumor cells involved in relapse. The first published pediatric cancer genomes showed that child tumors have ~10-20 fold fewer mutations than adult tumors, raising the possibility to characterize all involved signaling pathways separately or in proper combinations in a proper model system, e.g. stem or tumor initiating cells.

You recently attended the third Annual European Functional Genomics User’s Group Meeting in Cologne, Germany; which Sigma sponsors. What about the meeting did you find most interesting or helpful to your research?
There were several very interesting presentations, but I most liked the round table discussions between the presenters and the other participants. This gave the opportunity for trouble-shooting, setting up new cooperations, etc.

Having attended two of the three European User’s Group Meetings, why would you recommend the meeting to others?
The meetings give a terrific overview of new developments in gene knockdown technologies, both from a developer and a user perspective. I have recommended the meeting to people, and will continue to do so.

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Carla Grandori, M.D., Ph.D. Interview with Carla Grandori, M.D., Ph.D.
Director, Quellos High Throughput Screening Core
Affiliate Investigator Fred Hutchinson Cancer Research Center

Conducted July 2010

How did you come to work at the Quellos High Throughput Screening Core at the University of Washington?
In 2008, I was working at Rosetta, which was owned by Merck at the time. I was invited to be part of a planning committee to create the CORE because of my siRNA expertise. During the course of the committee meetings, I was asked to head the CORE. This really came a good time because I saw a real need for a HTSC in academia and was eager to bring this resource to the Seattle cancer research community. The fact was that nothing like this existed at the time and I could see a real need for the facility. Since I was able have my 50/50 position with Fred Hutchinson and still have my own lab, it really was a perfect fit.

Screening an siRNA or shRNA library is an involved process. What advice would give somebody who is interested in beginning the RNAi screening process?
Working with a screening facility is a great idea for a beginner. The automation at these facilities allows a researcher to screen accurately and rapidly, usually more so than "by hand." It also defrays the cost of having to purchase a library or gene set if you only need a few screens. Choosing the correct library or gene set is important as well. Personally, I prefer an siRNA library that is arrayed rather than pooled. It avoids the need for complicated deconvolution and it is more sensitive. I find it worth the extra cost versus a pooled library. Also, it enables the generation of data with statistical significance by performing screens with triplicate data points. Any beginning researcher should be sure to have good knowledge of the active molecular pathways, either by gene expression or genomic analysis in the cell lines intended to be used. Be prepared to use bioinformatics to analyze the resulting data, because the amount of data generated can be surprisingly large. Integration of the results with microarray and genomic data, will facilitate prioritization of the "hits."

What are the RNAi screening options out there for the average small and medium sized labs?
Doing focused screens with a sub-set of a library would allow a small lab to work with an arrayed system in a reasonable amount of time for a reasonable amount of money. What is important is choosing the correct genes, cells and correct sub sets for your work.

As the Director of a High Throughput Screening Core, what kind of screens do you see most often?
We are seeing a lot of siRNA screens with viability or reporter read outs. We have done few with high content readouts as well. In my opinion, these high content screens are going to be big in the future.

What is the next "big thing" in RNAi?
I think that the arrayed lentiviral libraries and high content screens are going to be big. As the prices of these will be more affordable, more and more people can start making use of the technology. The main advantage is that lentiviral libraries will bypass the need for transfection optimization of siRNAs. While a pooled library is less expensive, it is the arrayed libraries that really let you get to the heart of what is going on. We'll be seeing more and more people following up on hit from siRNA screens with shRNA clones, I think. Finally, as we are a long way from the use of RNAi as a therapeutic, I believe that screening for small molecule inhibitors to the identified targets will continue be needed to generate targeted drugs.

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Rick Cohen, Ph.D. Interview of Rick Cohen, Ph.D.
Assistant Research Professor at Keck Center for Collaborative Neuroscience and Director of the Rutgers University Stem Cell Core Facility

Conducted June 2010

How did you come to work at be the director of the Stem Cell Research Center at Rutgers?
I was offered a position at Rutgers in the newly built Stem Cell Research Center by Dr. Wise Young and Marty Grumet. We applied for new State funding initiatives and received a nearly 3 million dollar grant to establish a program project and core in hESC biology. As the only one with previous experience with hESCs, I spearheaded the Core Facility activities, whereas other senior faculty focused on their research projects using hESCs.

You are known for your work with Rutgers’ Stem Cell Training course. How did you move from research to this? What kind of help was available to Scientists interested in Stem Cell Research prior to the course being available?
In my previous position, Mr. Ed Satell sponsored a collaboration with the Technion, specifically with the laboratory of Dr. Joseph Itskovitz, a pioneer in the hESC field. Part of the collaboration was to set up a training course in NJ. At that time, Dr. Itskovitz collaborated with Dr. Mahendra Rao, and carried out a course at Johns Hopkins, with the help of his senior research Dr. Michal Amit. She along with another lab member came to my previous lab at Coriell to help run the first course, which was sponsored by a NJ State grant from the Commission on Science and Technology. After the first course, I moved to Rutgers, and with the continued support of Mr. Ed Satell, the State Grant, and several generous vendors, We ran several more training courses. To date, close to 100 scientists from Academia, and Industry, from NJ, and all over the world have been trained. Before I ran my course there were a few courses sponsored by NIH, however they have discontinued funding for this type of training because they feel enough labs are available to “self-train”. Likewise, due to budget shortfalls, NJ has discontinued funding for all stem cell research, which has put this highly successful program into question. We felt that NJ needed continued training opportunities, as many graduate students needed this specialized help. In addition, many Pharmaceutical companies (in NJ) were getting into hESC or IPSC research, and needed their personnel to learn the basics on how to maintain stem cell cultures. It made sense to continue this program, but a lack of funding may force us to close our doors. Ironically, this year we were asked to publish our course as a handbook, and will do so in early 2011. Since we constantly update the techniques, we feel that an evolving training course is needed to keep the field moving forward.

Could you describe some of your experiences working with shRNA in stem cells? What are some of their applications in regenerative medicine?
We have had some unexpected results using shRNA in hESCs. I have to admit, you learn a lot about your gene and development when knocking something down in hESCs. It works, but doesn’t always give you the expected results. We knocked down RhoA, and while it was clear some shRNA clones worked better than others, lowering RhoA in hESCs also lowered pluripotency genes. We found out that a 50% or so knock-down allowed us to still culture hESCs, however greater knock-downs made it impossible to cultivate the cells as hESCs! I’m not sure we really understand the role of shRNA yet, however others in our lab center are looking at knocking down genes directly in injured spinal cord to help limit the endogenous inhibitors of regeneration. Still, an injured nervous system is complex, and it may take years before we understand the roles of single genes in a complex system. Currently, the senior faculty in our center, Drs. Marty Grumet, Dr. Melitta Schachner, and Ron Hart are looking deeply into to roles of shRNA, and even microRNA in Spinal Cord Injury.

What are some of the implication of being able to target specific genes in stem cells? How do you see this fitting into the future of human health?
Targeting single genes allows use to question their function. In stem cells it allows us to question their role. These are two different things, like comparing the function of a car, versus the role of a car in the life of a race car driver. I’m not sure we can really fathom the lesson to be learned from knocking out single genes in hESCs, but if we study their role on development, differentiation and cell division we can learn a tremendous amount about how our cells misbehave. Clearly the relation of hESCs and cancer are clear, so lessons learned can be applied to future understanding of different types of cancer. Further, understanding the complexities of cell division, like the investigations of my Core Facility/Course co-director, Dr. Jennifer Moore, will help understand how to be able to transplant stem cell safely without the fear of tumor formation.

What is the next “big thing” in Stem Cell biology?
This is hard to predict. Many say personalized medicine, but it’s highly impractical to create personalized libraries of stem cells due to the cost. I think if we can elucidate the “transplantibility” of a stem cell product, this would propel hESC research back into the lime-light. Right now we don’t know if this field is a great tool for discovery, like drug-discovery and testing, or will it yield a marketable health resource for the future. However, it’s clear the race for stem cells is into a field that produces a GMP grade transplantable product. Right now, Dr. Wise Young, the Director of the Center, is working on Cord Blood stem cells for treatment of Spinal Cord Injury. It’s a little different than hESCs, however, the lessons learned from this type of project could help others move hESC derived products into the clinical setting. In the past, my collaborators at the Technion produced human pacemaker cells from hESCs, which were functionally incorporated into a pig (heart) model. Clearly they were able to create a biological pacemaker, which is just one early finding in this field. I’m sure they are interested in a human trial, which would provide a “permanent” solution for many people with heart disease. Others at the Technion, and around the world, look at producing Islet cells for Insulin production. Still we don’t know how to safely transplant them for a long term cure. All in good time.

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