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Updated: May 11, 2025
Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts
Published on: December 17, 2016
Protocol for assessing regulatory elements in murine heart using an AAV9-based massively parallel reporter assay
Kaiyu Wang1, Meng Meng2, Minghong Leng2
1Department of Pharmacology, Joint Laboratory of Guangdong-Hong Kong Universities for Vascular Homeostasis and Diseases, School of Medicine, Southern University of Science and Technology, Shenzhen 518055, Guangdong Province, China; Guangdong Cardiovascular Institute, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510080, China.
This study introduces a new method using adeno-associated virus serotype 9 (AAV9) and massively parallel reporter assays (MPRA) to measure regulatory element activity in mouse hearts. This protocol helps quantify enhancer function in vivo.
Area of Science:
- Genomics
- Cardiovascular Biology
- Molecular Biology
Background:
- The mammalian genome has thousands of regulatory elements sensitive to stimuli.
- Understanding these elements is crucial for deciphering gene regulation in cardiac function.
Purpose of the Study:
- To present a protocol for assessing regulatory element function in the murine cardiac system.
- To enable in vivo quantification of enhancer activity using a novel MPRA approach.
Main Methods:
- Utilizing an adeno-associated virus serotype 9 (AAV9)-based massively parallel reporter assay (MPRA).
- Describing steps for enhancer candidate selection, MPRA library construction, and animal delivery.
- Detailing sequencing library generation and data analysis pipeline for in vivo studies.
Main Results:
- The developed protocol offers a comprehensive workflow for enhancer activity assessment.
- The method allows for quantitative analysis of regulatory element function in the heart.
Conclusions:
- This AAV9-MPRA protocol provides a robust method for studying cardiac regulatory elements in vivo.
- The workflow facilitates a deeper understanding of gene regulation in the mammalian heart.
