# Advanced Recombinase Technology for Tissue Engineering Applications > **NIH NIH F31** · BOSTON UNIVERSITY (CHARLES RIVER CAMPUS) · 2020 · $35,829 ## Abstract PROJECT SUMMARY/ABSTRACT Site-specific recombinases (SSRs) have been used for decades to study the effects of gene knockout and cell lineage tracing. More recently, they have shown a strong capacity for logic computation in complex synthetic gene networks, or circuits. The utility of SSRs lies in their ability to permanently recombine DNA sequences, flanked by recombination sites, in response to an input stimulus. At present, their use in defining cell types or reporting on gene expression is limited to a small group of highly phenotype-specific promoters that function in a digital (on or off) manner in target cell types. Many phenotypes, though, cannot be easily defined or isolated based on the expression of a single gene; a much more generalizable strategy would use a combinatorial approach to report on the expression of multiple non-specific genes. The recent advent of split, chemically dimerizable SSRs has greatly expanded the number of genes that may be reported on simultaneously using a small number of SSRs. However, in order to use these effectively to define complex phenotypes, it is necessary to characterize SSR activity in response to different levels of gene expression, directly related to promoter strength. Additionally, we will need a mechanism for tuning SSR activity independent of promoter activity. This characterization strategy and tool development will provide a much broader range of useful genes/promoters for multiplexed and spatiotemporal interrogation of cell phenotypes, rather than relying on those exhibiting a digital on or off state. Recent efforts in standardizing the measurement and characterization of synthetic biology ‘parts’ has allowed for the collection of quantitative data to create a set of design rules for their use. Leveraging these, the goal of this work is to define a framework for the study of SSR dynamics in response to variable promoter activity, demonstrate the ability to tune SSR activity in response to expression level, and validate these technologies to enhance the controlled expression of SSRs to isolate complex cell phenotypes. First, I will expand the existing split recombinase library by creating self-dimerizing SSRs for use in cell lineage tracing and drug screening. Next, I will define and further develop and demonstrate a framework for studying SSRs in response to promoter strength and the effect of tuning SSR expression independent of promoter activity. Finally, I will demonstrate the scientific benefit of these tools by using split Cre and Flp SSRs to identify, isolate and characterize atrial and ventricular cardiomyocytes from a mixed population. In this third aim, I will use promoters known to be high or low expressing in each cell type, but are not useful in the current digital identification approach due to off-target basal activity. With this work, I aim shift the paradigm of SSR circuit development from the creation of one-off devices to a modular, plug and play system akin to the facile... ## Key facts - **NIH application ID:** 9989633 - **Project number:** 5F31HL149334-02 - **Recipient organization:** BOSTON UNIVERSITY (CHARLES RIVER CAMPUS) - **Principal Investigator:** Justin Henry Letendre - **Activity code:** F31 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2020 - **Award amount:** $35,829 - **Award type:** 5 - **Project period:** 2019-09-01 → 2022-08-31 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/9989633 ## Citation > US National Institutes of Health, RePORTER application 9989633, Advanced Recombinase Technology for Tissue Engineering Applications (5F31HL149334-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9989633. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*