Studies on Idiotype-driven T-B collaboration (copy)
Aim: to study the biological processes involved in T cell-B cell collaboration which is an important part of adaptive immunity, especially of antibody production. This application builds on FOTS ID 13707, which will expire this year.
Expected distress: the experiments are not aimed at modelling disease, rather to study principles of basic biology.
Distress will be related to routine experimental procedures, such as anesthesia, i.v. or i.p. injections and saphenous vein blood sampling. We have now mouse strains that express fluorescent proteins (eCFP and dsRed) since 2017 (mice have already been included in FOTS ID13707). FP are non-toxic and non-immunogenic. The sequences are derived from normal living organisms. Their use is widespread in molecular biology. The transgenes are introduced into the founder mouse genomes using homologous DNA recombination. We have by now gained some experience on mice that express these proteins and we have not recorded any clinical symptoms, nor did we notice tissue changes.
Expected Scientific and societal benefit: to increase our understanding relating to the biological principles of T-B collaboration, B cell maturation, memory formation and antibody production. Furthermore, as some of these processes contribute to the development of autoimmune diseases, results drawn from the experiments will increase our understanding of autoimmune disease. The use of the fluorescent strains will enable imaging modalities and allow us to extract more relevant biological information from each mouse.
Number of animals needed: we will use transgenic and knock-in mice (several strains, please see in the appropriate section). All mice are maintained at our facility (KPM-OUS, Rikshospitalet). We aim to use a maximum of 2346 animals in four years.
The 3 R's: we will use in vitro experiments to complement our studies, where possible. In in vitro experiments, interacting B and T or B and B cells will be first purified from the spleens and lymph nodes of donor mice and mixed in tissue culture wells. This will reduce, to some extent the amount of mice used, since less donor mice are needed. However, this may not be performed using all strains, since some have very low frequencies of the cells of interest (which however makes them more physiological). We will reduce the number of experimental animals to a minimum. Improvement: we will rely largely on the experience we obtained during the course of previous experiments. These were developed under FOTSID 13707, but also other protocols. We know how many B cells, T cells and BCR ligand are needed for a productive response, for example. The use of fluorescent proteins enable potential modes of imaging by the IVIS system, which also relate to typing procedures, thereby reducing stress related to blood sampling for some of the strains.
Expected distress: the experiments are not aimed at modelling disease, rather to study principles of basic biology.
Distress will be related to routine experimental procedures, such as anesthesia, i.v. or i.p. injections and saphenous vein blood sampling. We have now mouse strains that express fluorescent proteins (eCFP and dsRed) since 2017 (mice have already been included in FOTS ID13707). FP are non-toxic and non-immunogenic. The sequences are derived from normal living organisms. Their use is widespread in molecular biology. The transgenes are introduced into the founder mouse genomes using homologous DNA recombination. We have by now gained some experience on mice that express these proteins and we have not recorded any clinical symptoms, nor did we notice tissue changes.
Expected Scientific and societal benefit: to increase our understanding relating to the biological principles of T-B collaboration, B cell maturation, memory formation and antibody production. Furthermore, as some of these processes contribute to the development of autoimmune diseases, results drawn from the experiments will increase our understanding of autoimmune disease. The use of the fluorescent strains will enable imaging modalities and allow us to extract more relevant biological information from each mouse.
Number of animals needed: we will use transgenic and knock-in mice (several strains, please see in the appropriate section). All mice are maintained at our facility (KPM-OUS, Rikshospitalet). We aim to use a maximum of 2346 animals in four years.
The 3 R's: we will use in vitro experiments to complement our studies, where possible. In in vitro experiments, interacting B and T or B and B cells will be first purified from the spleens and lymph nodes of donor mice and mixed in tissue culture wells. This will reduce, to some extent the amount of mice used, since less donor mice are needed. However, this may not be performed using all strains, since some have very low frequencies of the cells of interest (which however makes them more physiological). We will reduce the number of experimental animals to a minimum. Improvement: we will rely largely on the experience we obtained during the course of previous experiments. These were developed under FOTSID 13707, but also other protocols. We know how many B cells, T cells and BCR ligand are needed for a productive response, for example. The use of fluorescent proteins enable potential modes of imaging by the IVIS system, which also relate to typing procedures, thereby reducing stress related to blood sampling for some of the strains.