TY - JOUR A1 - Schoetz, U. A1 - Deliolanis, N.C. A1 - Ng, D. A1 - Pauli, Jutta A1 - Resch-Genger, Ute A1 - Kühn, E. A1 - Heuer, S. A1 - Beisker, W. A1 - Köster, R.W. A1 - Zitzelsberger, H. A1 - Caldwell, R.B. T1 - Usefulness of a Darwinian system in a biotechnological application: evolution of optical window fluorescent protein variants under selective pressure T2 - PLoS one N2 - With rare exceptions, natural evolution is an extremely slow process. One particularly striking exception in the case of protein evolution is in the natural production of antibodies. Developing B cells activate and diversify their immunoglobulin (Ig) genes by recombination, gene conversion (GC) and somatic hypermutation (SHM). Iterative cycles of hypermutation and selection continue until antibodies of high antigen binding specificity emerge (affinity maturation). The avian B cell line DT40, a cell line which is highly amenable to genetic manipulation and exhibits a high rate of targeted integration, utilizes both GC and SHM. Targeting the DT40's diversification machinery onto transgenes of interest inserted into the Ig loci and coupling selective pressure based on the desired outcome mimics evolution. Here we further demonstrate the usefulness of this platform technology by selectively pressuring a large shift in the spectral properties of the fluorescent protein eqFP615 into the highly stable and advanced optical imaging expediting fluorescent protein Amrose. The method is advantageous as it is time and cost effective and no prior knowledge of the outcome protein's structure is necessary. Amrose was evolved to have high excitation at 633 nm and excitation/emission into the far-red, which is optimal for whole-body and deep tissue imaging as we demonstrate in the zebrafish and mouse model. PB - PubMed Central KW - Fluorescent protein KW - Quantum yield PY - 2014 UR - https://opus4.kobv.de/opus4-bam/frontdoor/index/index/docId/31976 AN - OPUS4-31976 SN - 1932-6203 VL - 9 IS - 9 SP - e107069-1 EP - e107069-12 AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany