Reassessing Tcf21-iCre for Adult Cardiac Fibroblast Gene Deletion: Limited Recombination Efficiency Following Tamoxifen Injections

Abstract

Highlights What are the main findings? Tcf21-iCre demonstrates minimal recombination efficiency in adult cardiac fibroblasts after tamoxifen injection, with mono-allelic events indicating low Cre activity. Neither pro-fibrotic stress nor additional tamoxifen dosing improves recombination efficiency. What are the implications of the main findings? Tcf21-iCre cannot reliably achieve broad gene deletion in adult cardiac fibroblasts with i.p. tamoxifen protocols. The field requires alternative genetic tools to advance antifibrotic therapeutic strategies targeting adult fibroblasts. Abstract Cardiac fibrosis plays a crucial role in heart failure, but research in this field remains challenging due to heterogeneity of cardiac fibroblast populations. In the past years, several Cre-drivers have been proposed for cardiac fibroblast-specific deletion of floxed genes in adult mouse research models. Here, we evaluated whether Tcf21-iCre, a tamoxifen-inducible Cre recombinase (iCre) under the endogenous Tcf21 promoter, was able to successfully target two floxed loci (Hypoxia inducible factor 1a (Hif1a) and the fluorescent reporter locus mTmG) after intraperitoneal tamoxifen injections. Six-week-old mice received intraperitoneal tamoxifen injections on five consecutive days to induce Cre activity, and the recombination efficiency was measured in isolated fibroblasts via qPCR, Western Blot and fluorescence microscopy. Recombination efficiency was very low, with minimal reduction in expression of Hif1a. For the mTmG locus, both bi-allelic and mono-allelic recombination was detected, with mono-allelic events suggesting low Cre activity even within Tcf21-expressing fibroblasts. The addition of experimental models of heart failure with reduced or preserved ejection fraction as pro-fibrotic stimuli or an inclusion of an additional round or higher doses of tamoxifen injections did not improve recombination efficiency of the targeted alleles. Thus, Tcf21-iCre targets only a small proportion of fibrogenic fibroblasts following i.p. tamoxifen injection, limiting its usability in loss-of-function studies that require broad fibroblast coverage and highlighting the need for alternative Cre-drivers or delivery strategies to facilitate antifibrotic therapy development.