title: Integrated non-invasive system for quantifying secreted human therapeutic hIL2.
abstract: Cell encapsulation has been used to treat diabetes, amyotrophic lateral sclerosis, and other chronic ailments by the secretion of therapeutic proteins in vivo. Detection of these proteins typically requires invasive procedures such as blood sampling or device extraction, however. In this article, a non-invasive means of measuring secreted protein concentration using a co-expressed red fluorescent protein marker is developed. A bicistronic expression vector was constructed for the intracellular production of a red fluorescent protein marker and the secreted production of human interleukin-2 (hIL2). The destabilized red fluorescent protein, DsExDR, was selected for its rapid turnover, as well as its ability to emit red light, which is readily transmitted through mammalian tissue. Transfections of this bicistronic vector into three cell lines C2C12, HEK293, and Jurkat showed linear correlations between the expressed proteins, DsExDR (intracellular) and hIL2 (secreted), with transfection DNA concentration. Correspondingly, there was a linear correlation between secreted product (hIL2) and intracellular marker (DsExDR). As transfection DNA was increased, Jurkat cells were found to increase secreted hIL2 in direct proportion to the accumulated DsExDR. HEK293 and C2C12 cells expressed and secreted significantly more hIL2 than the Jurkat cells, while still maintaining a linear relationship. Thus, all three cell lines were suitable hosts for the bicistronic expression of DsExDR and expression and secretion of therapeutic hIL2. This reporting strategy may find the greatest use in cell encapsulation therapy.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, Maryland, USA.
authors.forename: Chong Wing
authors.initials: CW
authors.lastname: Yung
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Barbari
authors.forename: William E
authors.initials: WE
authors.lastname: Bentley
mesh_terms: D000818:Animals; D001187:Artificial Organs; D015870:Gene Expression / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics* / Q000276:immunology; D005822:Genetic Vectors; D006801:Humans; D007376:Interleukin-2 / Q000032:analysis / Q000378:metabolism* / Q000627:therapeutic use; D019169:Jurkat Cells; D007668:Kidney / Q000166:cytology; D008164:Luminescent Proteins / Q000032:analysis / Q000493:pharmacokinetics; D051379:Mice; D011994:Recombinant Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism* / Q000627:therapeutic use; D014162:Transfection / Q000379:methods*; D000097573:Red Fluorescent Protein
chemical_list: D007376:Interleukin-2; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D000097573:Red Fluorescent Protein; C508594:IL2 protein, human