Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
SHR Neuro Krebs Kardio Lipid Stoffw Microb
Wakula, P; Bisping, E; Kockskamper, J; Post, H; Brauer, S; Deuter, M; Oehlmann, R; Besenfelder, U; Lai, FA; Brem, G; Pieske, B.
CMV promoter is inadequate for expression of mutant human RyR2 in transgenic rabbits
J Pharmacol Toxicol Methods. 2010; 63(2):180-185
Doi: 10.1016/j.vascn.2010.09.007
Web of Science
PubMed
FullText
FullText_MUG
- Führende Autor*innen der Med Uni Graz
- Pieske Burkert Mathias
- Wakula-Heinzel Paulina
- Co-Autor*innen der Med Uni Graz
- Bisping Egbert Hubertus
- Kockskämper Jens
- Post Heiner
- Altmetrics:
- Dimensions Citations:
- Plum Analytics:
- Scite (citation analytics):
- Abstract:
- Introduction: Fundamental differences in Ca(2+) 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. (C) 2010 Elsevier Inc. All rights reserved.
- Find related publications in this database (using NLM MeSH Indexing)
- Animals -
- Animals, Genetically Modified - genetics
- Cytomegalovirus - genetics
- Gene Expression -
- Green Fluorescent Proteins - biosynthesis Green Fluorescent Proteins - chemistry Green Fluorescent Proteins - genetics
- Humans -
- Mice -
- Mutation -
- Myocardium - metabolism
- Promoter Regions, Genetic -
- RNA, Messenger - biosynthesis RNA, Messenger - genetics
- Rabbits - blood Rabbits - genetics
- Recombinant Fusion Proteins - biosynthesis Recombinant Fusion Proteins - chemistry Recombinant Fusion Proteins - genetics
- Reverse Transcriptase Polymerase Chain Reaction - methods
- Ryanodine Receptor Calcium Release Channel - biosynthesis Ryanodine Receptor Calcium Release Channel - genetics
- Tachycardia, Ventricular - genetics Tachycardia, Ventricular - metabolism
- Transgenes -
- Find related publications in this database (Keywords)
- CMV promoter
- Ryanodine receptor 2 (RyR2)
- Transgenic rabbits
- Methods