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;