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.
Department of Molecular Genetics and Microbiology, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854, USA.
A L
AL
Pacchia
M E
ME
Adelson
M
M
Kaul
Y
Y
Ron
J P
JP
Dougherty
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