Objective—To use transient and stable transfection of
Chinese hamster ovary cells to clone the gene encoding
feline erythropoietin (feEPO) protein, characterize
the expressed protein, and assess its biological activity.
Sample Population—Cultures of Chinese hamster
ovary or TF-1 cells.
Procedure—The gene encoding feEPO was cloned
into a eukaryotic expression plasmid. Chinese hamster
ovary cells were transiently or stably transfected
with the plasmid. Expressed recombinant feEPO
(rfeEPO) protein was purified from transiently transfected
cells. The protein was characterized by use of
SDS gel electrophoresis and western blot analysis.
Biological activity was assessed by measuring thymidine
incorporation by TF-1 erythroleukemic cells.
Results—Purified rfeEPO from supernatants of transiently
transfected cells was determined to be 34 to
40 kilodaltons (kd) by use of SDS gel electrophoresis,
whereas the molecular weight predicted from the
amino acid sequence was 21.5 kd. The banding pattern
and high molecular weight suggested the protein
was glycosylated. The rfeEPO proteins derived from
transient or stable transfections subsequently were
determined to be biologically active in vitro.
Conclusions and Clinical Relevance—The gene
encoding feEPO can be transfected into eukaryotic
cells, and the expressed rfeEPO protein is biologically
active in vitro. Cats with chronic renal failure often
are anemic as a result of reduced expression of
erythropoietin (EPO). Treatment with human-derived
EPO stimulates RBCs in anemic cats; however, treatment
is often limited by the development of antibodies
directed against the recombinant human protein,
which can then cross-react with endogenous feEPO.
Recombinant feEPO may prove beneficial for use in
cats with chronic renal failure. (Am J Vet Res 2003;
Objective—To identify biomarker proteins for B-cell lymphoma in canine serum by use of surface-enhanced laser desorption-ionization time-of-flight (SELDI-TOF) mass spectrometry and build classification trees with multiple biomarkers that have high sensitivity and specificity for that tumor type.
Sample Population—Sera from 29 dogs with B-cell lymphoma and 87 control dogs (approx equal numbers of healthy dogs, dogs with malignant cancers other than B-cell lymphoma, and dogs with various nonneoplastic diseases or conditions).
Procedures—Serum samples were fractionated chromatographically and analyzed via SELDI-TOF mass spectrometry. Peak amplitudes of the spectra from the 2 sample groups were compared to identify potential biomarker peaks, and classification trees were built by use of computer software to detect patterns formed by multiple biomarkers among SELDI data sets.
Results—Several biomarker protein peaks in canine serum were identified, and a classification tree was built on the basis of 3 biomarker protein peaks. With 10-fold cross-validation of the sample set, the best individual serum biomarker peak had 75% sensitivity and 86% specificity and the classification tree had 97% sensitivity and 91% specificity for the classification of B-cell lymphoma.
Conclusions and Clinical Relevance—On the basis of biomarker proteins identified in canine serum, classification trees were constructed, which may be useful for the development of a diagnostic test for B-cell lymphoma in dogs. Further investigation is needed to determine whether these biomarkers are useful for screening susceptible dog populations or for monitoring disease status during treatment and remission of B-cell lymphoma in dogs.