Mature insulin production by engineered non-beta cells.
OBJECTIVE
To pursue insulin and islet-transplantation replacement therapy for type 1 diabetes based on engineered human non-beta cells which secrete mature insulin.


METHODS
Human proinsulin cDNA was cloned from its genomic gene and mutated by overlap extension PCR, introducing furin consensus cleavage sequences (Arg-Xaa-Lys/Arg-Arg). An expression vector encoding a genetically modified human proinsulin cDNA was generated and transduced to Hela, 293, and L02 cells by lipofectin-mediated DNA transfection. Following G418 screening, the surviving L02 cells were selected and enriched. Insulin levels in the supernatant and cells were evaluated using radioimmunoassay and immunofluorescence staining.


RESULTS
Three sites in the insulin gene were mutated simultaneously. Insulin gene modified cells were able to express insulin at different levels: 8.45 - 188.00 microIU/24 h/2.0 x 10(6) Hela cells and 159.88 - 242.14 microIU/24 h/2.0 x 10(6) 293 cells for transient expression, and 2.56 - 61.95 microIU/24 h/2.0 x 10(6) from several L02 clones screened with G418. No insulin was released by control cells. Furthermore, immunofluorescence staining confirmed that proinsulin was stored as vacuoles in the cytoplasm of L02 cells.


CONCLUSIONS
A correctly mutated human proinsulin cDNA was obtained successfully, transfected and expressed efficiently in non-beta cells, lending support to the study of somatic gene therapy in diabetes mellitus.
Shen
Kuntang
K
Department of General Surgery, Zhongshan Hospital, Fudan University, Shanghai 200032, China.
Qin
Xinyu
X
Xiao
Huasheng
H
Zhang
Xin
X
Xu
Xiangru
X
Han
Zeguang
Z
D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000235:genetics; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007328:Insulin / Q000235:genetics / Q000378:metabolism*; D011384:Proinsulin / Q000235:genetics*; D011863:Radioimmunoassay; D014162:Transfection
D018076:DNA, Complementary; D007328:Insulin; D011384:Proinsulin