Adenovirus mediated gene transduction of primarily isolated mouse islets.
Expansion of pancreatic islet cell populations, especially the beta cells, using a currently available ex vivo gene transfer technology is important to develop cell therapies to treat Type I diabetes. In this study, we evaluated adenovirus mediated gene transfer efficiency in primarily isolated mouse islet cells in two types of culture conditions: freshly isolated suspended islets and cultured islets with monolayer formation. A recombinant replication deficient adenovirus vector encoding a green fluorescence protein (GFP) cDNA, Ad/ CMV-GFP, was used in the present transduction experiments. Rat 804G derived extracellular matrix (804G-ECM) and 3-isobutyl-1-methylxanthine (IBMX) were used to facilitate monolayer formation of the isolated mouse pancreatic islets. Suspended islets were transfected with Ad/CMV-GFP at more than 95% efficiency. However, analysis of immunohistochemical stains for insulin and glucagon in thin sliced sections of the islets revealed that GFP expression was localized just in the outer cells of the islets, almost all of which were glucagon positive alpha-cells. The beta-cells existing at the inner area of the suspended islets were GFP negative. In contrast, under the condition of the islets in monolayer formation cultures, all of the islet cells including the beta-cells were efficiently infected with Ad/CMV-GFP. To achieve an efficient adenoviral gene transfer to the pancreatic beta-cells, monolayer formation of the islets is critical. Such culture conditions were facilitated by combining 804G-ECM with IBMX.
Narushima
Michiki
M
Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan.
Okitsu
Teru
T
Miki
Atsushi
A
Yong
Chen
C
Kobayashi
Kazuya
K
Yonekawa
Yukihide
Y
Tanaka
Kimiaki
K
Ikeda
Hideaki
H
Matsumoto
Shinichi
S
Tanaka
Noriaki
N
Kobayashi
Naoya
N
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics; D005109:Extracellular Matrix; D005822:Genetic Vectors; D005934:Glucagon / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006652:Histological Techniques; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology / Q000502:physiology; D016381:Islets of Langerhans Transplantation; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D016513:Mice, SCID; D051381:Rats; D014018:Tissue Distribution; D014161:Transduction, Genetic / Q000379:methods
D007328:Insulin; D049452:Green Fluorescent Proteins; D005934:Glucagon; D015056:1-Methyl-3-isobutylxanthine