Specific targeting of pancreatic islet cells in vivo by insulin-promoter-driven adenoviral conjugated reporter genes.
BACKGROUND
The objective of this study was to determine whether rat insulin promoter (RIP) could, in a mouse model, direct expression of an adenovirus-mediated reporter gene specifically into pancreatic islets via systemic delivery.


METHODS
Five hundred and eight base pairs of the RIP DNA sequence were constructed into an adenoviral vector containing a lacZ reporter gene (Adeno-RIP-lacZ). The cytomegalovirus (CMV) promoter was constructed to drive lacZ reporter-gene expression (Adeno-CMV-lacZ) and used as controls. In vitro transient transfection assays were performed to determine levels of reporter-gene expression and compared with that of liposome-mediated plasmid transfection. SCID mice were bred and housed in the barrier BL-4 animal facility. At 2 months of age, the human pancreatic cancer cell PANC-1 was intraperitoneally injected into male mice. Two months after the tumor cell inoculation, mice were injected with 10(7) adenoviral particles via tail veins. After gene delivery, mice were sacrificed at different time points to determine transgene expression levels. Complete necropsies were performed. Morphological alterations were determined using hematoxylin and eosin (H&E) staining, and distribution of the reporter lacZ gene was determined by immunohistochemistry analyses.


RESULTS
Adenoviral-driven reporter-gene expression resulted in more than 5 times higher transgene expression compared with conventional plasmid transfections. In Adeno-RIP-lacZ-injected mice, lacZ expression was specifically detected in pancreatic islets. By contrast, in Adeno-CMV-lacZ-injected mice, lacZ gene expression was observed in multiple organs and tissues. Mononuclear cell infiltration and liver cell inflammation were found in Adeno-CMV-lacZ-treated mice. Similar phenomena were observed in islet cells of Adeno-RIP-lacZ-treated mice. A significantly higher level of reporter-gene expression was also found at the edge of in-vivo-inoculated human pancreatic tumors.


CONCLUSIONS
These results demonstrate that RIP-directed reporter-gene expression was found specifically in mouse pancreatic islets and implanted human pancreatic cancer cells. These data thus demonstrate that the combination of an adenoviral vector and a tissue-specific promoter could lead to an enhanced and more specific transgene expression in vivo.
Wang
Xiaoping
X
Michael E. DeBakey Department of Surgery, Baylor College of Medicine, 1709 Dryden, Suit 1500, Houston, TX 77030, USA.
Olmsted-Davis
Elizabeth
E
Davis
Alan
A
Liu
Shihe
S
Li
Zhijun
Z
Yang
Jie
J
Brunicardi
F Charles
FC
D000256:Adenoviridae; D000818:Animals; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D015870:Gene Expression; D018390:Gene Targeting / Q000379:methods*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D007328:Insulin; D007515:Islets of Langerhans; D007763:Lac Operon; D008297:Male; D051379:Mice; D016513:Mice, SCID; D009368:Neoplasm Transplantation; D010190:Pancreatic Neoplasms / Q000628:therapy; D011401:Promoter Regions, Genetic; D051381:Rats
D007328:Insulin