Year: 2009
Type: article
Abstract: Ribozymes have emerged as promising therapeutics for HIV and have been shown to bind and cleave target RNA's in a sequence-specific manner.The HIV-1 regulatory protein tat plays an essential role in the upregulation of viral transcription and elongation of viral transcripts, and has therefore been identified as a target for ribozyme studies.To further study the use of these reagents, we have designed a number of hammerhead ribozymes targeted to specific sequences within the HIV-1 NL43 tat sequence.One of these, Tat5910 and its non-catalytic control Tat5910∆, were cloned into the retroviral vector pSuper.retro.neo+GFP(pSRNG) for cell culture studies.Recombinant retrovirus particles were generated by transient transfection of 293T cells using a two-plasmid system consisting of the helper plasmid, pPAM3, and pSRNG5910∆.Identical experiments were carried out using two Mo-MLV retroviral vectors, pLNCE, and pLNCLZRz.The resulting recombinant virus was used to transduce NIH-3T3 cells, and virus titer (virus particles/ml) was determined from the number of Green Fluorescent Protein (GFP)-or β-galactosidase-positive cells.Although producer cells transfected with pSRNG5910∆ expressed GFP, the transfection efficieny was low.This resulted in levels of transduced NIH-3T3 cells that were too low to obtain a reliable titer measurement.Attempts to optimize titer by increasing the number of transfected producer 293T cells was successful using pLNCE and pLNCLZRz.Positive GFP or β-galactosidase expression in NIH-3T3 cells transduced with LNCE and LNCLzRz recombinant virus indicated that our two-plasmid virus production system was successful.Extremely low titer along with lower cellular expression of GFP suggests that the pSRNG5910 vectors may be better suited to virus production using a stable producer cell line such as PA317
Authors: Lindsey E. Padgett, William H. Jackson
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2992094608