Ruth Popa-Wagner

Home Institution
University of Heidelberg

Host Institution
Center for Gene Therapeutics, McMaster University, Hamilton, Ontario, Canada

E-Mail:
ruth@ruthwagner.de

Research Topic
see Abstract
Personal Reactions to the Canadian Experience
Being in Canada now for six months, I can say that the Canadian way of life and the extremely welcoming and helpful people around me have had a great impact on my scientific progress as well as on my personal development. I have been integrated into the group from the first moment on and everybody showed good will in helping and training me. Especially my supervisor Jonathan Bramson, who always answered my questions with interest and enthusiasm and Katja Linher, who trained and taught me about molecular cloning techniques, gave me new ideas and expanded my scientific horizon. During my free time I had the opportunity to go on canoeing-, camping- and dogsledding-trips with the university's outdoor club, where I met great people and made very good friends! Those outdoor trips not only tied personal bonds but also gave me the opportunity to experience Canada's amazing nature. Coming to Canada was a very important step in my life!
Greatest Difficulties Encountered
As it is very easy to get a visa and a work permit for Canada, I did not have to deal with those obstacles. Finding an accommodation was also not too difficult, so the only thing that took a while getting used to was living in a student house with six guys and trying to accept their way of keeping the house clean.
Most humorous incident
I cannot think of many days without laughing and giggling with either my colleagues or my non-lab friends, so it is pretty hard to point out one single humorous incident. But I think one of the most hilarious events while being here happened on the dogsledding trip. Two brothers were riding the sled, one was sitting in the basket of the sled, and the other one was standing on the vats, trying to keep their dog team under control. Suddenly the dogs started running very fast and the guy standing on the vats fell off, but his brother, who was sitting in the basket, didn't notice. So the dogs kept running without driver and the guy who fell off had to run even faster to try to catch up with them. And I can tell you, running in the deep snow and trying to be faster than a 6-dog team is not fun at all!

Helpful Hints for Future Students

  • If you decide to come to Canada you don't have to worry too much about getting a work permit. You just have to submit the forms on time, which is about four weeks prior to departure.
  • If you are in Canada during wintertime don't miss to go on a dogsledding trip! This was one of the greatest experiences of my life!
  • "Germans drink a lot of beer". Be prepared to hear this saying more than once!

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Abstract on Research Topic
Molecular cloning of third-generation lentiviral vectors and generation of chimeric mouse models by lentiviral transduction of murine bone marrow cells

Author:
Ruth Popa-Wagner (not published)

Institution:
Center for Gene Therapeutics, McMaster University in Hamilton, Ontario, Canada

Background:
Lentiviruses are a family of complex retroviruses inducing chronic and progressive diseases. Especially in the field of gene therapy HIV-1-based lentiviruses attracted much attention for their ability to stably transduce non-dividing cells such as hematopoietic stem cells. As lentiviruses stably integrate into the host's chromosomes, they maintain a long-term expression of the transgene which is required for efficient gene therapy approaches. In addition, they do not transfer viral genes, avoiding the destruction of transduced cells by cytotoxic T cells. Lentiviral vectors have a relatively large cloning capacity, making them suitable for subcloning of large transgenes which also qualifies them for high-potency gene delivery tools.

Purpose and Methods:
The overall goal of this project is to generate chimeric mouse models, carrying the primate diphtheria toxin receptor (pDTR) within their hematopoietic stem cells. Administration of diphtheria toxin to those mice will allow us to deplete subsets of cells within the hematopoietic system in order to study the regulation between the different subsets. We are especially interested in studying the regulation of CD4+ and CD8+ T lymphocytes within the hematopoietic environment in order to better understand adaptive immune responses. To generate the chimeric mouse model, murine, nondividing hematopoietic stem cells will be transduced with transgenic lentiviruses carrying the gene for the pDTR along with a selectable marker. Lethally irradiated recipient C57Bl/6 mice will then be rescued by adoptive transfer of the transduced hematopoietic stem cells. The objective of this project is to genetically engineer lentiviral vectors expressing the pDTR along with a selectable marker under the control of the EMCV-IRES. Lentiviruses generated by cotransfection of 293FT cells with lentiviral vectors and packaging constructs will be used for generating chimeric mouse models in order to study T lymphocyte regulation in vivo by conditional cell ablation.

Results:
The recombinant lentiviral vectors, carrying the gene for the diphtheria toxin receptor along with a selectable marker (T cell coreceptor CD8 and CD4, respectively) were constructed successfully. The protocol for generating the Lentiviruses has to be improved further as viral titers are not yet satisfactory. Cells that have been infected with lentivirus could be detected by flow cytometry. Preliminary data showed that lentiviruses were capable of integrating into the genome of nonproliferating murine hematopoietic stem cells.