Introduction
The department of Medicinal Chemistry at the Vrije Universiteit in Amsterdam, the lab where I’ve been doing my PhD studies, has been focusing on signaling pathways activated by human as well as viral chemokine receptors. Chemokine receptors control the immune response but also play a role in cancer. A few years before I started my PhD, my department showed that the viral chemokine receptor US28 encoded by the human cytomegalovirus (HCMV) is constitutively active, meaning it is signaling in a ligand-independent manner (Casarosa et al., 2001, JBC). Since then, a significant number of papers have taken interest in that viral receptor protein as it is suspected to have some implication in pathological conditions induced by HCMV. The goal of my PhD is to determine which signaling pathways are activated by US28 and whether it contributes to processes in cancer or atherosclerosis. I showed during the first phase of my PhD studies that US28 is indeed activating proliferative mechanisms and angiogenic events which eventually lead to the formation of tumors in a xenograft tumor model in nude mice. This resulted in a nice paper in the Proceedings National Academy of Sciences (Maussang et al., 2006, PNAS).

Wanna make a mouse in New York?
Similarly to HCMV, the Kaposi sarcoma associated herpes virus (KSHV) encodes a constitutively active chemokine receptor, refers to as ORF74, which possesses oncogenic properties. Dr Sergio Lira was a pioneer in the creation of transgenic mice expressing this viral oncogene, and during a visit at the Vrije Universiteit, my supervisor agreed with him that the next logical step for us would be to create a transgenic mouse expressing US28.
At that stage, I had not been involved in this discussion of the project and was not aware they were thinking of sending someone from our lab to New York. A few months later, during the FIGON Dutch Pharmacological Days, my supervisor approached me and casually asked me “Do you want to go to New York to work with US28 transgenic mice?”. It didn’t take me too long to figure out that it was an incredible opportunity for me to experience this completely new field and broaden my scientific perspectives.

The Immunology Institute, Mount Sinai School of Medicine, New York
In the Immunology Institute in the Mount Sinai School of Medicine, Dr Lira studies the role of chemokines and cytokines in a variety of biological processes including leukocyte trafficking, lymphoid neogenesis, angiogenesis, and cancer. Work in his laboratory has defined an important role for CC, CXC, and CX3C chemokines in homeostasis and disease, and has provided a rational for development of novel therapeutic agents for the treatment of inflammatory conditions. He also studies chemokine and cytokine-like molecules encoded by viruses to understand their role in viral pathogenesis and immune evasion.
As an example, to better understand the role of chemokine receptors in angiogenesis Dr Lira has studied the KSHV-encoded chemokine receptor ORF74. This viral receptor displays high constitutive signaling activity that is positively regulated by angiogenic CXC chemokines. He showed that transgenic expression of ORF74 is sufficient to induce an angioproliferative disease in mice that closely resembles Kaposi’s sarcoma. To better understand these mechanisms underlying ORF74 function, he developed conditional models for ORF74 expression in mice. Using these models he showed that ORF74 induces tumor formation by autocrine and paracrine mechanisms. Furthermore, he has shown that ORF74 is a critical molecule for disease induction; elimination of ORF74 expression during the angioproliferative phase induces regression of the lesions.

Let’s go to the mouse room
My labwork in Amsterdam primarily consists of cell culture and in vitro assays. So, when I arrived in my lab in New York where all the experiments are performed on mice or with extracted tissues, I had to first learn a lot about the mouse anatomy. Luckily, my colleagues were very helpful and very patient with my lack of knowledge about how a mouse “looks like from the inside”, and they taught me everything that had to be known to be able to work by myself. Although at the beginning it seemed a bit difficult to handle the mice and localize all the different organs I had to work with, I quickly realized that it was just like putting pieces of a puzzle together, all parts are always at the same place and look the same. The first time I went to “see the mice” was quite an experience. All our mice were kept in the animal facility, located in the basement of our research building and access was restricted to a limited number of scientists. The first step was to wear the appropriate labcoat, but also overshoes, a hairnet, a mask and gloves. Once “dressed up”, we were ready to access the transgenic mice room. I didn’t really know what to expect in this “mouse room”, but to my surprise, mice cages were stacked in huge racks. All seemed to have different cards in front of them, different colors and it all looked very confusing. With time, I learnt that everything was extremely well organized, labeled and followed precise directives for the good functioning of the lab.

The projects
Another point I realized in New York was that working with living creatures takes much longer to give results than simple cells in culture. The aim of the project was to create transgenic mice that express the HCMV-encoded GPCR US28. To me, that sounded very easy, but to achieve such a result, quite a few rounds of mice pregnancies, matings, genotyping by PCR, phenotyping had to take place. Basically, after the injection of the plasmid containing US28 gene into the nuclei of fertilized mice eggs, we got several founder mice, which are the mice used to create lines of transgenic animals that will be used to study the protein of interest in vivo. After we established the lines of mice, we obtained some interesting initial findings and pursued the project. With further characterizations, this could lead to a nice publication. Having the opportunity to work with mice was incredibly interesting and it opened a broad new range of ideas and hypothesis that would not have occurred to me by simply working with in vitro systems.

New York, New York…
The first days in New York were quite overwhelming. Beside losing all my habits I had in that “big city” of Rotterdam and experiencing floods in the streets of New York straight away after landing, I felt a little lost. But within a few days, connections with post-docs from the lab and other departments from Mount Sinai established and I got the chance to discover nice aspects of the city, the big sights from the movies, nice places to go out, and the very enjoyable Central Park. I was living one “block” away from Central Park and describing the Park as the lungs of the city is really true. This huge green open space gives a more human dimension to the city and especially during the summer, everyone migrates and gathers in Central Park and spend days lying in the sun. No need to count the numerous picnic parties that happened there, the free concerts and afternoons spent with friends. Summer in general in New York was amazing as although temperature and humidity make you enjoy very much the air conditioning anywhere you can find it, the city organizes lots of events in the different “boroughs” of the city. This also allowed me to discover places in the City I would have never seen otherwise and my summer months in New York will remain some of the best moments.

To be continued…
After the initial characterization of the transgenic mice, it was already time for me to go back to my lab in Amsterdam. I am very happy and grateful to have initiated this project and to have the opportunity to work with in vivo models. Now, a post-doc from my lab in Amsterdam has taken over the projects and will finalize them. With this experience, I learnt a great deal about mice, in vivo work and new techniques that I would have never thought of using during my PhD in Amsterdam. I would like to thank the Zwanenberg Organization to have supported me during this incredible opportunity in New York and given me the chance to widely broaden my scientific horizon.