Method for production of high titer virus and high efficiency retroviral mediated transduction of mammalian cells

A method for transducing with high efficiency mammalian target cells with foreign genes, said method comprising:

• A) culturing stably transfected 293 cells producing replication-defective recombinant retroviral vectors carrying a selected foreign gene;
• B) separating said transfected 293 cells from supernatant;
• and
• C) exposing said mammalian target cells to said supernatant, whereby target cells efficiently transduced with said foreign gene are obtained.

A method for transducing with high efficiency mammalian target cells with foreign genes, said method comprising:

• A) culturing transfected human cells producing replication-defective recombinant retroviral vectors carrying a selected foreign gene;
• B) separating said transfected human cells from supernatant;
• and
• C) exposing said mammalian target cells to said supernatant, whereby target cells efficiently transduced with said foreign gene are obtained.

A method for transducing mammalian target cells with foreign genes, said method comprising:

• A) transiently transfecting a first population of mammalian cells that produce virus, wherein the first population of mammalian cells are stably transfected with at least two retroviral packaging plasmids, which are transfected sequentially, comprising retroviral helper DNA sequences derived from a replication-incompetent retroviral genome encoding in trans all virion proteins required for packaging a replication-incompetent retroviral vector at high titer, without the production of replication-competent helper virus, said retroviral helper DNA sequence lacking the region encoding the native enhancer and/or promoter of the viral 5' LTR of said virus and lacking both the psi function sequence responsible for packaging helper genome and the 3' LTR, and encoding a foreign enhancer and/or promoter functional in a selected mammalian cell, and including an SV40 polyadenylation site with
• a retroviral vector encoding a foreign gene to produce replication-defective recombinant retroviral vectors carrying said foreign gene in said first population of mammalian cells;
• and
• B) cocultivating said first population of mammalian cells producing replication-defective recombinant retroviral vectors carrying said foreign gene with a second population of mammalian target cells, wherein said recombinant retroviral vectors transduce said second population of target cells with said foreign gene.

A method for transducing mammalian target cells with foreign genes, said method comprising:

• A) transiently transfecting a first population of mammalian cells that produce virus, wherein the first population of mammalian cells are stably transfected with at least two retroviral packaging plasmids, which are transfected sequentially, comprising retroviral helper DNA sequences derived from a replication-incompetent retroviral genome encoding in trans all virion proteins required for packaging a replication-incompetent retroviral vector at high titer, without the production of replication-competent helper virus, said retroviral helper DNA sequence lacking the region encoding the native enhancer and/or promoter of the viral 5' LTR of said virus and lacking both the psi function sequence responsible for packaging helper genome and the 3' LTR, and encoding a foreign enhancer and/or promoter functional in a selected mammalian cell, and including an SV40 polyadenylation site with
• a retroviral vector encoding a foreign gene to produce replication-defective recombinant retroviral vectors carrying said foreign gene in said first population of mammalian cells;
• and
• B) separating said first population of mammalian cells from supernatant containing recombinant retroviral vectors;
• and
• C) incubating said supernatant with a second population of mammalian target cells, wherein said recombinant retroviral vector transduces said second population of target cells with said foreign gene.