Rapid and highly efficient inducible cardiac gene knockout in adult mice using AAV-mediated expression of Cre recombinase

Abstract

Aims: Inducible gene targeting in mice using the Cre/LoxP system has become a valuable tool to analyse the roles of specific genes in the adult heart. However, the commonly used Myh6-MerCreMer system requires time-consuming breeding schedules and is potentially associated with cardiac side effects, which may result in transient cardiac dysfunction. The aim of our study was to establish a rapid and simple system for cardiac gene inactivation in conditional knockout mice by gene transfer of a Cre recombinase gene using adeno-associated viral vectors of serotype 9 (AAV9).

Methods and results: AAV9 vectors expressing Cre under the control of a human cardiac troponin T promoter (AAV-TnT-Cre) enabled a highly efficient Cre/LoxP switching in cardiomyocytes 2 weeks after injection into 5- to 6-week-old ROSA26-LacZ reporter mice. Recombination efficiency was at least as high as observed with the Myh6-MerCreMer system. No adverse side effects were detected upon application of AAV-TnT-Cre. As proof of principle, we studied AAV-TnT-Cre in a conditional knockout model (Srf-flex1 mice) to deplete the myocardium of the transcription factor serum response factor (SRF). Four weeks after AAV-TnT-Cre injection, a strong decrease in the cardiac expression of SRF mRNA and protein was observed. Furthermore, mice developed a severe cardiac dysfunction with increased interstitial fibrosis in accordance with the central role of SRF for the expression of contractile and calcium trafficking proteins in the heart.

Conclusions: AAV9-mediated expression of Cre is a promising approach for rapid and efficient conditional cardiac gene knockout in adult mice.

Keywords: Adeno-associated virus; Cardiomyopathy; Conditional transgenic mouse; Gene regulation; Serum response factor.