CMV promoter is inadequate for expression of mutant human RyR2 in transgenic rabbits.
BACKGROUND
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.


CONCLUSIONS
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.
Wakula
Paulina
P
Department of Cardiology, Medical University of Graz, Auenbruggerplatz 15, 8036 Graz, Austria.
Bisping
Egbert
E
Kockskämper
Jens
J
Post
Heiner
H
Brauer
Sabine
S
Deuter
Marion
M
Oehlmann
Ralph
R
Besenfelder
Urban
U
Lai
F Anthony
FA
Brem
Gottfried
G
Pieske
Burkert
B
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D003587:Cytomegalovirus / Q000235:genetics*; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D006801:Humans; D051379:Mice; D009154:Mutation; D009206:Myocardium / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011817:Rabbits / Q000097:blood / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D019837:Ryanodine Receptor Calcium Release Channel / Q000096:biosynthesis* / Q000235:genetics*; D017180:Tachycardia, Ventricular / Q000235:genetics / Q000378:metabolism; D019076:Transgenes; D000098850:Polymorphic Catecholaminergic Ventricular Tachycardia
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins