Construction of Recombinant Adeno-Associated Virus Vector Containing the Rat Preproinsulin II Gene
| Autor: | Scott T. Cooper, Rahul M. Jindal, Matthew M. Burton, Lan Peng, Richard A. Sidner, Markian R. Bochan |
|---|---|
| Rok vydání: | 1997 |
| Předmět: |
Preproinsulin
Transcription Genetic viruses Genetic Vectors Radioimmunoassay Adenocarcinoma Biology medicine.disease_cause Polymerase Chain Reaction Transduction (genetics) Plasmid Tumor Cells Cultured medicine Animals Humans Insulin Protein Precursors Adeno-associated virus Gene Southern blot Recombination Genetic Rous sarcoma virus Gene Transfer Techniques Virion Dependovirus biology.organism_classification Virology Molecular biology Rats Pancreatic Neoplasms Restriction enzyme Genes Surgery Plasmids Proinsulin |
| Zdroj: | Journal of Surgical Research. 69:193-198 |
| ISSN: | 0022-4804 |
| Popis: | We have investigated a possible delivery system for the rat preproinsulin II gene ( rI 2 ) utilizing a recombinant adeno-associated virus (rAAV) vector system, with the long-term goal of engineering stably infected insulin-producing cell lines. The rAAV vector was chosen because it is a safe and nonpathogenic method for gene transfer. The plasmid pBC12BI (ATCC) was purified and digested with restriction enzymes SspI and StuI to release a fragment containing the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter-driven rat preproinsulin II gene ( rI 2 ). Subsequently, the RSV- rI 2 gene fragment was cloned into the Bam HI site of rAAV vector plasmid pWP-19 to produce the rI 2 recombinant plasmid designated pLP-1. The pWP-19 also encodes the AAV inverted terminal repeats for integration and replication and the herpes virus thymidine kinase promoter-driven gene for neomycin resistance (neo R ). The cell line 293 (ATCC) was then co-transfected with pLP-1 and helper plasmid pAAV\AD, which is required for viral replication. The rAAV genome, now containing rI 2 , was rescued using adenovirus and packaged into mature AAV virions termed vLP-1. Finally, human pancreatic adenocarcinoma cells (HPAC; ATCC) were exposed to vLP-1, selected for G418 resistance, and screened for insulin production. Successful rescue was confirmed by Southern blot analysis using the rI 2 gene probe derived from the original plasmid. The final titer of 1.25 × 10 9 particles/ml was determined by DNA slot blots using pLP-1 as the standard. HPAC cells were infected with vLP-1 (termed HPAC/ rI 2 ). Integration of the rI 2 genome in G418-resistant clones was confirmed by Southern blot analysis and again after 6 months in culture by amplification of the rI 2 gene by PCR. Insulin gene transcription was confirmed by RT-PCR. We have developed a rAAV-mediated gene transfer system for the rat preproinsulin II gene. Successful transduction and stable integration of rI 2 into HPAC was achieved. Production of insulin by HPAC/ rI 2 was confirmed by RIA and RT-PCR, validating this system as an effective approach to experimental gene therapy. |
| Databáze: | OpenAIRE |
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