Next generation marker-based vector concepts for rapid and unambiguous identification of single and double homozygous transgenic organisms.

For diploid model organisms, the actual transgenesis processes require subsequent periods of transgene management, which are challenging in emerging model organisms due to the lack of suitable methodology. We used the red flour beetle Tribolium castaneum, a stored-grain pest, to perform a comprehensive functional evaluation of our AClashOfStrings (ACOS) and the combined AGameOfClones/AClashOfStrings (AGOC/ACOS) vector concepts, which use four clearly distinguishable markers to provide full visual control over up to two independent transgenes. We achieved comprehensive statistical validation of our approach by systematically creating seventeen novel single and double homozygous sublines intended for fluorescence live imaging, including several sublines in which the microtubule cytoskeleton is labeled. During the mating procedures, we genotyped more than 20,000 individuals in less than 80 working hours, which corresponds to about 10 to 15 s per individual. We also confirm the functionality of our combined concept in two double transgene special cases, i.e. integration of both transgenes in close proximity on the same chromosome and integration of one transgene on the X allosome. Finally, we discuss our vector concepts regarding performance, genotyping accuracy, throughput, resource saving potential, fluorescent protein choice, modularity, adaptation to other diploid model organisms and expansion capability.

Simultaneous Live Imaging of Multiple Insect Embryos in Sample Chamber-Based Light Sheet Fluorescence Microscopes.

Light sheet-based fluorescence microscopy offers efficient solutions to study complex processes on multiple biologically relevant scales. Sample chamber-based setups, which are specifically designed to preserve the three-dimensional integrity of the specimen and usually feature sample rotation, are the best choice in developmental biology. For instance, they have been used to document the entire embryonic morphogenesis of the fruit fly Drosophila melanogaster and the red flour beetle Tribolium castaneum. However, many available live imaging protocols provide only experimental frameworks for single embryos. Especially for comparative studies, such approaches are inconvenient, since sequentially imaged specimens are affected by ambient variance. Further, this limits the number of specimens that can be assayed within a given time. We provide an experimental framework for simultaneous live imaging that increases the throughput in sample chamber-based setups and thus ensures similar ambient conditions for all specimens. Firstly, we provide a calibration guideline for light sheet fluorescence microscopes. Secondly, we propose a mounting method for multiple embryos that is compatible with sample rotation. Thirdly, we provide exemplary three-dimensional live imaging datasets of Drosophila, for which we juxtapose three transgenic lines with fluorescently labeled nuclei, as well as of Tribolium, for which we compare the performance of three transgenic sublines that carry the same transgene, but at different genomic locations. Our protocol is specifically designed for comparative studies as it pro-actively addresses ambient variance, which is always present in sequential live imaging. This is especially important for quantitative analyses and characterization of aberrational phenotypes, which result e.g., from knockout experiments. Further, it increases the overall throughput, which is highly convenient when access to light sheet fluorescence microscopes is limited. Finally, the proposed mounting method can be adapted for other insect species and further model organisms, e.g., zebrafish, with basically no optimization effort.