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Cloning and sequencing of the canine and feline cardiac troponin I genes

Mark Rishniw BVSc, MS1, Stephen C. Barr BVSc, PhD2, Kenny W. Simpson BVMS, PhD3, Nena J. Winand DVM, PhD4, and Joyce A.M. Wootton PhD5
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  • 1 Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY.
  • | 2 Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY.
  • | 3 Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY.
  • | 4 Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, NY.
  • | 5 Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY.

Abstract

Objective—To determine the gene sequences of canine and feline cardiac troponin I (cTnI), express the protein from the cloned gene in vitro, and validate the use of a commercial cTnI serum analyzer in these species via detection of the expressed protein or comparison of sequence homology.

Sample Population—Samples of ventricular myocardium from 5 healthy adult mixed-breed dogs and 5 healthy adult domestic shorthair cats.

Procedure—The RNA was extracted from myocardial samples, and cDNA was synthesized via reverse transcriptase polymerase chain reaction and sequenced. The canine cDNA for the coding region was expressed in cell culture and analyzed by western blot and sandwich enzyme-linked immunosorbent assays.

Results—Canine and feline cTnI genes were cloned and sequenced. Homology of the nucleotide and amino acid sequences of the canine and feline cTnI genes with human and rodent cTnI genes were high; the greatest homology was detected between canine and feline genes (95% and 96%, respectively). Recombinant canine cTnI protein was detected by a commercial serum cTnI analyzer and by western blot analysis.

Conclusions and Clinical Relevance—Results indicated that commercial cTnI analyzers can be used to measure serum cTnI concentration from dogs and cats. Additionally, our preliminary characterization of the feline cTnI gene may facilitate further investigation of cTnI and its role in familial hypertrophic cardiomyopathy in cats. ( Am J Vet Res 2004;65:53–58)

Abstract

Objective—To determine the gene sequences of canine and feline cardiac troponin I (cTnI), express the protein from the cloned gene in vitro, and validate the use of a commercial cTnI serum analyzer in these species via detection of the expressed protein or comparison of sequence homology.

Sample Population—Samples of ventricular myocardium from 5 healthy adult mixed-breed dogs and 5 healthy adult domestic shorthair cats.

Procedure—The RNA was extracted from myocardial samples, and cDNA was synthesized via reverse transcriptase polymerase chain reaction and sequenced. The canine cDNA for the coding region was expressed in cell culture and analyzed by western blot and sandwich enzyme-linked immunosorbent assays.

Results—Canine and feline cTnI genes were cloned and sequenced. Homology of the nucleotide and amino acid sequences of the canine and feline cTnI genes with human and rodent cTnI genes were high; the greatest homology was detected between canine and feline genes (95% and 96%, respectively). Recombinant canine cTnI protein was detected by a commercial serum cTnI analyzer and by western blot analysis.

Conclusions and Clinical Relevance—Results indicated that commercial cTnI analyzers can be used to measure serum cTnI concentration from dogs and cats. Additionally, our preliminary characterization of the feline cTnI gene may facilitate further investigation of cTnI and its role in familial hypertrophic cardiomyopathy in cats. ( Am J Vet Res 2004;65:53–58)