Journal List > J Korean Diabetes Assoc > v.31(1) > 1062487

Kim, Moon, Ko, Ahn, Song, Lim, Lee, Yoo, Son, Yoon, Cha, Son, Yoon Kim, and Han: Glucose-dependent Insulin Secretion from Genetically Engineered K-cells Using EBV-based Episomal Vector

Abstract

Background

Type 1 diabetes mellitus is an autoimmune disease resulting in destruction of the pancreatic beta cells. Insulin gene therapy for these patients has been vigorously researched. The strategy for achieving glucose-dependent insulin secretion in gene therapy relies on glucose-responsive transcription of insulin mRNA and the constitutive secretory pathway of target non-beta cells. We observed that genetically engineered K-cells using Epstein-Barr virus (EBV)-based episomal vector can produce glucose-regulated insulin production.

Methods

Green fluorescent protein (GFP) or rat-preproinsulin (PPI) expression cassette transcriptionally controlled by the promoter of glucose dependent insulinotropic peptide (GIPP) is fused to pCEP4 containing the origin of replication (oriP) and Epstein-Barr virus nuclear antigen 1 (EBNA-1). CMV promoter was replaced by subcloning the GIPP into pCEP4 to generate pGIPP/CEP4. Two recombinant EBV-based episomal vectors, pGIPP/GFP/CEP4 and pGIPP/PPI/CEP4, were constructed. pGIPP/GFP/CEP4 and pGIPP/PPI/CEP4 containing K-cell specific GIPP were co-transfected into STC-1. K-cell was isolated from the clonal expansion of the fluorescent cells selected by hygromycin treatment in STC-1, and were analyzed for the expression of glucokinase (GK) or transcription factors involved in pancreas development. K-cells concurrently transfected with pGIPP/PPI/CEP4 and pGIPP/GFP/CEP4 were analyzed for the transcripts of PPI by RT-PCR, and for the glucose dependent insulin expression by immunocytochemistry or insulin assay using ultra-sensitive rat-specific insulin ELISA kit.

Result

STC-1 was stably-transfected with pGIPP/GFP/CEP4 along with pGIPP/PPI/CEP4. Genetically selected fluorescent K-cells expressed GK and transcription factors involved in pancreas development. And K-cells transfected with pGIPP/PPI/CEP4 contained detectable levels of PPI transcripts and showed glucose-dependent immunoreactive insulin secretion.

Conclusion

We identified genetically engineered K-cells which exert a glucose-dependent insulin expression using EBV-based episomal vector. The similarities between K-cells and pancreatic beta cells support that K-cells may make effective and ideal targeting cells for insulin gene therapy or alternative cell therapy.

Figures and Tables

Fig. 1
Schematic representation of the cloning steps employed to produce the pGIPP/GFP/CEP4 vector.
(A) EBV-based plasmid (pCEP4) vector containing the EBV nuclear antigen 1 (EBNA-1) and the origin of replication (oriP). CMV promoter excised as BglII/NotI was replaced by rat GIP promoter (GIPP) which was digested with BamHI and NotI from pGIPP/CR2.1. Then, GFP fragment excised with XhoI from pGFP/EGFP-C2 was fused to the XhoI restriction site of pGIPP/CEP4 to generate pGIPP/GFP/CEP4. (B) Linear structure of the pGIPP/GFP/CEP4 showing the BglII/BamHI cloning site.
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Fig. 2
Schematic representation for the construction of insulin gene expression cassettes.
(A) Rat preproinsulin (PPI) cDNA amplified by the primer set including BamHI and XhoI site was incorporated into the pBluescript or pGEX vector. From the pPPI/Bluescript vector, PPI was excised as a NotI/XhoI and purified by agarose gel electrophoresis. Then PPI fragment was fused to the NotI/XhoI restriction site of the pGIPP/CEP4 vector to generate pGIPP/PPI/CEP4. (B), (C) Linear structure of the pGIPP/PPI/CEP4 and pPPI/GEX vector, respectively.
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Fig. 3
Confirmation of cloned vector.
A, PCR analysis of rat GIPP from pGIPP/CR2.1 vector; B, PCR analysis of rat PPI from pPPI/CR2.1 vector. GIPP or PPI gene is shown well incorporated into pCR2.1 TOPO Vector; C, pGIPP/PPI/CEP4 digested with NotI and SalI; D, Cloned expression vector of pGIPP/GFP/CEP4 digested with XhoI and SalI respectively. GIPP or PPI, and GIPP or GFP were digested well, and the nucleotide sequences were confirmed by sequence analysis (data not shown).
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Fig. 4
Expression of insulin from pPPI/GEX vector in Escherichia coli.
Western blot analysis of insulin from Escherichia coli expressing GST only (lanes 1, 2), negative control (lanes 3, 4), GST-tagged insulin (lanes 5, 6), and bead binding GST-tagged insulin (lane 7) were separated on 15% (w/v) SDS PAGE. Arrows indicate the induced GST only (lane 1-2) and GST-tagged insulin (lane 5-7). GST or rat-insulin antibody recognized a GST or GST-tagged insulin. A, lysates prior to IPTG induction; B, lysates after IPTG induction.
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Fig. 5
Isolation of K-cell from STC-1 cell line.
A, Schematic diagram of the isolation steps employed to separation from the K-cells; B, Fluorescence microscopic findings of the isolated K-cells from STC-1. Phase-contrast (I), fluorescence (II), and merge (III) micrographs of the STC-1 cells co-transfected with pGIPP/GFP/CEP4 and pGIPP/PPI/CEP4. Transfected cells with pGIPP/GFP/CEP4 emit green fluorescence (1), transfected cells formed a clone (2), and continuously subcultured cells (K-cells) emit green fluorescence (3).
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Fig. 6
Western blot analysis and RT-PCR analysis in K-cell.
A, Western blot analysis of glucokinase (GK) expression in K-cell and STC-1. Bovine aortic endothelial cell (BAEC) was used as a negative control and MIN6 was used as a positive control. More GK is expressed in K-cells than in STC-1, but not in BAEC; B, RT-PCR analysis of transcription factors related to pancreas development and β-cell functions represent K-cell is comparable to mouse islet cells. Nkx 6.1 and ngn3 were not expressed in K-cells. Pax4 was in K-cell but not in mouse islet cells. Lane MW: 2 ug 1-kb ladder; lane 1: k-cells; lane 2: mouse islet cells.
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Fig. 7
Insulin secretion analysis from K-cells.
A, RT-PCR for the selection of rat-preproinsulin mRNA in four different clones. K-cells transfected with pGIPP/PPI/CEP4 (in clone 4 only) expressed preproinsulin mRNA; B, RT-PCR analysis for preproinsulin mRNA from clone 4 cells in response to glucose. Samples were prepared either in the presence (+) or absence (-) of reverse transcriptase (RT); C, D, Immunocytochemical staining for insulin expression from the co-transfected with pGIPP/GFP/CEP4 and pGIPP/PPI/CEP4. Insulin expression showed glucose dependent manner. E, Effects of glucose on the insulin secretion from K-cells stably transfected with the pGIPP/PPI/CEP4 (clone 4 cells) compared with the pGIPP/GFP/CEP4 (clone 3 cells). Results are means ± SD from three independent triplicate experiments in each group.
*P < 0.01 vs clone 3.
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