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Updated: Jun 8, 2026
In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium
Published on: May 6, 2018
CMV promoter is inadequate for expression of mutant human RyR2 in transgenic rabbits
Paulina Wakula1, Egbert Bisping, Jens Kockskämper
1Department of Cardiology, Medical University of Graz, Auenbruggerplatz 15, 8036 Graz, Austria.
Introduction:
Fundamental differences in Ca²+ homeostasis between mice and larger mammals require the validation of the mechanisms of arrhythmogenesis before translation into human pathophysiology. The purpose of this study was to create transgenic rabbits that express defective human cardiac ryanodine receptor (hRyR2) with a mutation (R4497C) causing a clinically relevant arrhythmogenic syndrome.
Methods:
The construct pcDNA3-EGFP-hRyR2-R4497C with the CMV promoter was used to generate transgenic rabbits. The founder animals were created by microinjection and identified by PCR with specific primers for the EGFP sequence. The copy number of the transgene was quantified by real-time PCR using genomic DNA from blood cells. mRNA expression of EGFP-hRyR2-R4497C was quantified using RT-PCR with specific primers for the RyR2 and EGFP sequence. Protein expression of the transgene in heart and non-cardiac tissues was determined using immunoblots with antibodies directed against EGFP and RyR2.
Results:
Real-time PCR in peripheral blood cells identified several rabbit lines with the construct integrated into their genome. Transcription levels of the transgene were low (Ct>30). On the protein level, neither EGFP nor hRyR2 R4497C was detected in either cardiac or non-cardiac tissue. A truncated gene product (3' end and central part of hRyR2 R4497C, but not EGFP) could be detected at the mRNA level in the heart.
Discussion:
Lack of significant protein expression of the EGFP-RyR2 R4497C gene construct despite successful incorporation into the genomic DNA is due to combination of at least two factors: low mRNA expression, and truncation of the transgene on the mRNA level. Our results suggest that the CMV promoter may not be well suited for creating transgenic rabbits.
Insights
Transgenic rabbits carrying a human cardiac ryanodine receptor mutation did not express the functional protein due to low mRNA levels and truncation. This highlights challenges in using the CMV promoter for large animal models in cardiac research.
Area of Science:
- Cardiovascular Research
- Genetics and Molecular Biology
- Animal Models
Background:
- Mammalian calcium (Ca²⁺) homeostasis differs, necessitating validation of arrhythmogenesis mechanisms in larger animals before human application.
- Transgenic animal models are crucial for studying inherited cardiac conditions and validating therapeutic targets.
Purpose of the Study:
- To create transgenic rabbits expressing a defective human cardiac ryanodine receptor (hRyR2) with the R4497C mutation.
- To establish a large animal model for studying clinically relevant arrhythmogenic syndromes.
Main Methods:
- Generation of transgenic rabbits via microinjection of the pcDNA3-EGFP-hRyR2-R4497C construct.
- Quantification of transgene copy number, mRNA expression (EGFP-hRyR2-R4497C), and protein expression (EGFP, hRyR2) using PCR and immunoblotting.
Main Results:
- Transgenic rabbits with integrated hRyR2 R4497C construct were identified.
- Low mRNA expression (Ct>30) and absence of detectable EGFP or full-length hRyR2 R4497C protein in cardiac and non-cardiac tissues.
- Detection of a truncated hRyR2 R4497C mRNA product in the heart, but not EGFP.
Conclusions:
- Lack of significant transgene protein expression is attributed to low mRNA levels and mRNA truncation.
- The CMV promoter may be unsuitable for generating transgenic rabbits for cardiac research.
- Further optimization of gene delivery and promoter selection is needed for large animal models.
