Integrated non-invasive system for quantifying secreted human therapeutic hIL2

• Published in: Biotechnology and bioengineering Vol. 95; no. 5; pp. 938 - 945
• Main Authors: Yung, Chong Wing, Barbari, Timothy A., Bentley, William E.
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 05.12.2006; Wiley; Wiley Subscription Services, Inc
• Subjects: Animals; Artificial Organs; bicistronic; Biological and medical sciences; Biotechnology; cell encapsulation; Cellular biology; Correlation analysis; Cytokines; Deoxyribonucleic acid; destabilized DsRed-Express; DNA; fluorescence; Fundamental and applied biological sciences. Psychology; Gene Expression - genetics; Genes, Reporter - genetics; Genes, Reporter - immunology; Genetic Vectors; human interleukin-2; Humans; Interleukin-2 - analysis; Interleukin-2 - metabolism; Interleukin-2 - therapeutic use; Jurkat Cells; Kidney - cytology; Luminescent Proteins - analysis; Luminescent Proteins - pharmacokinetics; Mice; Protein synthesis; Recombinant Proteins - analysis; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Recombinant Proteins - therapeutic use; Red Fluorescent Protein; Therapy; Transfection - methods; Encapsulation; Human; Drug; cell encapsulation; destabilized DsRed-Express; human interleukin-2; Non invasive method; Fluorescence; Integrated system; bicistronic; Quantitative analysis
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.21064

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. © 2006 Wiley Periodicals, Inc.