Genetic Counter Constructs for Developmental Biology

All multi-cellular organisms begin their life as a single cell. As this cell divides into two cells and continues to multiply into a population, certain cells undergo epigenetic changes that cause them to differentiate, ultimately resulting in the formation of germ layers, organs, and complete organ systems. Current approaches to control these processes (or part of these processes) often involve 3D printing of scaffolds, 3D printing of cells, and/or complex temporal media changes from a small stem cell population. For all of these approaches and other possible approaches, it would be convenient to have cells undergo differentiation, or the expression of surface proteins autonomously without extensive experimenter intervention and micro-managing. Here we describe recombinase-based genetic counter constructs that can count cell divisions and initiate the over-expression of specific genes at specific counts. These constructs are designed to count in a binary manner such that 2^n counts can be achieved with n recombinases and their reversal directionality factors (RDFs/Xis). Furthermore, the readout mechanism is designed such that a unique set of genes can be expressed at each individual count in the process. We have developed a software program to design these constructs automatically given a truth table of which genes should be expressed at which counts and another software to input a recombinase-base circuit and verify the recombination events of the design. We show orthogonality validation for a large set of serine recombinases in human cell lines and some preliminary data for using this counting system with fluorescence-based readouts in immortalized human cell lines (K6562/HEK293T) and human induced-pluripotent stem (iPS) cells. Our long term goal is to get this system fully functional in human iPS cells where the readouts could be transcription factors, surface binding proteins, or other developmental-biology relevant genes.