Construction of recombinant adeno-associated virus vector containing the rat preproinsulin II gene

Lan Peng, Richard A. Sidner, Markian R. Bochan, Matthew M. Burton, Scott T. Cooper, Rahul M. Jindal

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

We have investigated a possible delivery system for the rat preproinsulin II gene (rI2) 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 (rI2). Subsequently, the RSV-rI2 gene fragment was cloned into the BamHI site of rAAV vector plasmid pWP-19 to produce the rI2 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 cotransfected with pLP-1 and helper plasmid pAAV/AD, which is required for viral replication. The rAAV genome, now containing rI2, 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 rI2 gene probe derived from the original plasmid. The final titer of 1.25 x 109 particles/ml was determined by DNA slot blots using pLP-1 as the standard. HPAC cells were infected with vLP-1 (termed HPAC/rI2). Integration of the rI2 genome in G418-resistant clones was confirmed by Southern blot analysis and again after 6 months in culture by amplification of the rI2 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 rI2 into HPAC was achieved. Production of insulin by HPAC/rI2 was confirmed by RIA and RT-PCR, validating this system as an effective approach to experimental gene therapy.

Original languageEnglish (US)
Pages (from-to)193-198
Number of pages6
JournalJournal of Surgical Research
Volume69
Issue number1
DOIs
StatePublished - Apr 1997
Externally publishedYes

Fingerprint

Dependovirus
Genes
Plasmids
Terminal Repeat Sequences
Insulin
Southern Blotting
Polymerase Chain Reaction
Genome
preproinsulin
Rous sarcoma virus
Cell Line
Investigational Therapies
Neomycin
Thymidine Kinase
Gene Amplification
Virus Replication
Adenoviridae
Genetic Therapy
Virion
Insulin Resistance

ASJC Scopus subject areas

  • Surgery

Cite this

Peng, L., Sidner, R. A., Bochan, M. R., Burton, M. M., Cooper, S. T., & Jindal, R. M. (1997). Construction of recombinant adeno-associated virus vector containing the rat preproinsulin II gene. Journal of Surgical Research, 69(1), 193-198. https://doi.org/10.1006/jsre.1997.5079

Construction of recombinant adeno-associated virus vector containing the rat preproinsulin II gene. / Peng, Lan; Sidner, Richard A.; Bochan, Markian R.; Burton, Matthew M.; Cooper, Scott T.; Jindal, Rahul M.

In: Journal of Surgical Research, Vol. 69, No. 1, 04.1997, p. 193-198.

Research output: Contribution to journalArticle

Peng, L, Sidner, RA, Bochan, MR, Burton, MM, Cooper, ST & Jindal, RM 1997, 'Construction of recombinant adeno-associated virus vector containing the rat preproinsulin II gene', Journal of Surgical Research, vol. 69, no. 1, pp. 193-198. https://doi.org/10.1006/jsre.1997.5079
Peng, Lan ; Sidner, Richard A. ; Bochan, Markian R. ; Burton, Matthew M. ; Cooper, Scott T. ; Jindal, Rahul M. / Construction of recombinant adeno-associated virus vector containing the rat preproinsulin II gene. In: Journal of Surgical Research. 1997 ; Vol. 69, No. 1. pp. 193-198.
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