An inducible packaging cell system for safe, efficient lentiviral vector production in the absence of HIV-1 accessory proteins.
Lentiviral vectors based on human immunodeficiency virus type 1 (HIV-1) possess the ability to deliver exogenous genes to both dividing and nondividing cells and to subsequently establish a stable provirus in these target cells, which can allow long-term expression of the transferred gene. Herein we describe a stable packaging cell line that is devoid of HIV-1 tat, vif, vpr, vpu, and nef. In order to avoid any risk of cytotoxicity associated with constitutive expression of HIV-1 protease or the VSV-G envelope protein, transcription of the packaging and envelope constructs was tightly controlled by employing the ecdysone-inducible system. Using this cell line, we have been able to consistently generate concentrated pseudotyped vector virus stocks with titers in the range of 10(8) IU/ml, which can efficiently transduce actively dividing and growth-arrested cells in vitro. This novel packaging cell line for lentiviral vectors facilitates the production of high-titer virus stocks in the absence of replication-competent virus and provides us with an important tool for use in future gene transfer studies.
Pacchia
A L
AL
Department of Molecular Genetics and Microbiology, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854, USA.
Adelson
M E
ME
Kaul
M
M
Ron
Y
Y
Dougherty
J P
JP
D002460:Cell Line; D004440:Ecdysone / Q000494:pharmacology; D015696:Gene Products, tat / Q000172:deficiency / Q000235:genetics; D016346:Gene Products, vif / Q000172:deficiency / Q000235:genetics; D016336:Gene Products, vpr / Q000172:deficiency / Q000235:genetics; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D015497:HIV-1 / Q000235:genetics*; D054298:Human Immunodeficiency Virus Proteins; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics; D008562:Membrane Glycoproteins; D014161:Transduction, Genetic; D014162:Transfection; D014759:Viral Envelope Proteins / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics; D054334:Viral Regulatory and Accessory Proteins / Q000172:deficiency / Q000235:genetics; D014779:Virus Replication; D054322:tat Gene Products, Human Immunodeficiency Virus; D054320:vif Gene Products, Human Immunodeficiency Virus; D054325:vpr Gene Products, Human Immunodeficiency Virus; D000086082:Viroporin Proteins
D004440:Ecdysone; D015696:Gene Products, tat; D016346:Gene Products, vif; D016336:Gene Products, vpr; D049452:Green Fluorescent Proteins; D054298:Human Immunodeficiency Virus Proteins; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D054320:vif Gene Products, Human Immunodeficiency Virus; D054325:vpr Gene Products, Human Immunodeficiency Virus; C028391:G protein, vesicular stomatitis virus; C514859:vpu protein, Human immunodeficiency virus 1; D000086082:Viroporin Proteins