Yearbook 0405


Andreas Bonertz
Home Institution Ruprecht-Karls-Universität, Heidelberg Host Institution(s) Center for Cancer Research Massachusetts Institute of Technology E-Mail: andreas.bonertz@gmx.de
Research Topic see Abstract
Personal Reactions to the U.S. Experience I had been to the US before when I spent a High School Year in San Antonio, Texas. Yet, as I can tell now, it has been a completely different experience this time. As before in Texas, I really enjoyed my time in Massachusetts. However, although Boston certainly is a very American city, it still has a European flair. Boston is a very nice, impressive city with many diverse things to do. It offers a lot of pubs, good restaurants, theatres, and very good sport teams (although it is exceptionally hard to get tickets for football or baseball games.) I happened to be around in the final days before the presidential election took place and, just as many people that I spoke to, I was very excited and hoping for the best. It was not too hard for me to deal with the not so positive election results, since pretty much all the people in my lab were feeling the same way. It seemed to me that a lot was going wrong in this country. Especially in the news, criticism of America is perceived as very unpatriotic. Besides that, I had a great time and learned a lot, both in my lab and otherwise.
Greatest Difficulties Encountered Without any doubt, the most difficult problems came up before I even got to the US. Four weeks before my planned departure date from Germany, the Principal Investigator in my lab left MIT and I was told that I could not come to the lab. Luckily, I was able to contact my supervisor in the MIT lab and explain to him that this would put me in a really bad situation. He talked to the new PI in the lab that he would now join and I was accepted to join the new lab with the same project as before. However, now the visa process was delayed, and I had to rebook my flights in order to leave ten days later than I had planned. After I arrived in the US no major difficulties came up.
Most humorous incident Four weeks after my arrival, the MIT Center for Cancer Research had a retreat at a conference center in New Hampshire over the weekend. Everybody was looking forward to that, since the retreats are known to be a lot of fun, with parties at night. My lab mates told me not to buy any liquor in advance, as we would stop in New Hampshire because it is a lot cheaper there. We stopped with two cars and about ten people and went inside. At the checkout they asked for our IDs and I did not have my passport with me. They would not accept my German ID Card, my expired Texas Drivers license, my European Drivers license or anything else. Now they would not sell anything to anybody who was with me, so nobody got to buy anything. We had to get back in the car and drive to the next store. So do not forget your passport if you want to buy some good wine.
Helpful Hints for Future Students Get a customer rewards card at the supermarkets (e.g. Shaws, Stop&Shop, CVS) where you shop. There are a lot of special offers and you get many things much cheaper. If you have fast Internet access at home, it is very cheap to call home using VoIP-providers, such as Sipgate or Skype. MIT students get free access to the Museum of Science and the Museum of Fine Arts in Boston. It is well worth visiting them. As an MIT student you also have free access to the MIT fitness centers, including an Olympic size pool.
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Abstract on Research Topic
A luciferase based assay for the functional validation of siRNAs

Authors:
Andreas Bonertz, Peter Sandy

Institution:
Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA

Introduction:
RNA silencing or RNA interference has emerged as one of the most powerful tools for researchers to specifically silence single genes. The silencing process of RNAi, which targets mRNA sequences for degradation, is sequence specific, so that only the desired sequences are targeted. One major problem has been to validate how efficient the designed siRNA sequences are for silencing. Most commonly used methods to determine efficiency of silencing, like Western blot, Real-Time PCR, or RNase protection assays are very time consuming and it is difficult to test a great number of siRNAs for their knockdown efficiency. The luciferase-based assay described here makes it possible to test many sequences for knockdown efficiency in a relatively short amount of time.

Procedure and Results:
Two vectors were cotransfected into 293FT cells in a 96-well plate format. One vector was the lentiviral pll3.7 vector with a U6 promoter followed by the coding sequence for the desired short hairpin RNA (shRNA). Also on this vector is a CMV promoter followed by the coding sequence for GFP in order to determine the rate of transfection of 293FT cells. The second vector is a modified version of the psiCHECK2 vector. The vector contains a promoter followed by the coding sequence for Renilla (Renilla reniformis) luciferase and the cDNA sequence for the gene of interest, followed by a stop codon. The cDNA sequence had been cloned into the vector using the Gateway Technology. Transcription yields one mRNA coding for both, the Renilla luciferase and the gene of interest, separated by a translational stop codon. If translated, only the Renilla luciferase is expressed. The amount of protein in the cell, and thus the knockdown by the shRNA, can be easily assessed by the amount of Renilla luciferase activity.

When RNA interference occurs, the RNA induced silencing complex (RISC) containing the single-stranded RNA strand can bind and degrade the mRNA. No Renilla luciferase activity can be measured. On the same vector, a Thymidin-Kinase promoter is followed by the coding sequence for the Firefly (Photinus pyralis) luciferase. The activity of the Firefly luciferase can be measured and is used to normalize the measured Renilla luciferase activity. Genes targeted by shRNAs in this assay were Rac I, Shp2, Caspase 1, Caspase 2, Caspase 6, Caspase 7, Caspase 8, and Caspase 9. Different numbers of shRNAs were tested on these genes. The luciferase activities were measured and the corresponding protein levels were determined. To validate these results, DO.11 cells were infected with the same shRNAs that had been used in the luciferase assay. Lentivirus was used to infect these cells. Infected cells were sorted for GFP and a western blot was performed. The knockdown efficiency was similar to the luciferase results.

Discussion:
Through simple cloning of the desired gene's cDNA sequence into the psiCHECK2 vector, and expression of this vector in a cell line, an unlimited amount of shRNAs can be easily tested for knockdown efficiency against this gene. Although computational algorithms for designing siRNA or shRNA sequences have become much more reliable on designing functional siRNA sequences, in silicio design and prediction cannot completely replace the functional test of the sequences used for a knockdown. To determine if and to what extend a specific siRNA is actually effectively reducing the amount of the targeted protein and if this knockdown is the responsible factor for an observed phenotype, a functional validation of the shRNA is needed. Using this luciferase-based assay, it is possible to do this validation in a simple in vitro assay.