Olivier Danos and Richard C. Mulligan developed packaging cell lines for generating helper-free recombinant retroviruses with amphotropic and ecotropic host ranges. These cell lines, named ψ CRIP and ψ CRE, were created by sequentially introducing two mutant Moloney murine leukemia virus (Mo-MuLV) proviral genomes with complementary mutations in the gag-pol or env regions into NIH 3T3 cells. Both genomes lacked the ψ sequence necessary for efficient viral particle encapsidation and had altered 3' ends. The resulting cell lines produced high-titer recombinant retroviruses without transferring packaging functions or generating helper virus, even under conditions where other cell lines could. This makes them valuable for in vivo gene transfer studies, particularly for cell lineage analysis and human gene replacement therapies. The initial step in generating recombinant retroviruses for mammalian gene transfer involves introducing a suitable proviral DNA vector into fibroblastic cells that produce viral proteins for encapsidation. Previous packaging cell lines, such as ψ-2 and ψ-AM, had issues with transferring packaging functions and generating helper virus. To address this, Danos and Mulligan introduced additional mutations into the ψ- genome to reduce the likelihood of these events. However, the possibility of recombinational events still existed, as mutations could be corrected by recombination with a ψ+ vector genome. To further eliminate the problem of packaging-function transfer and helper virus generation, Danos and Mulligan created two modified Mo-MuLV genomes with deletions of the ψ sequence and 3' LTR, encoding either the gag-pol or env gene. These genomes were introduced into NIH 3T3 cells through two rounds of transfection and coselection with different selectable markers. The resulting cell lines, ψ CRIP and ψ CRE, were characterized for their efficiency and safety. ψ CRIP produced amphotropic retroviruses, while ψ CRE produced ecotropic retroviruses. The packaging capacity of ψ CRIP and ψ CRE was tested using a mobilization assay, which showed that these cell lines did not transfer the ψ- genome or generate helper virus. Long-term cultivation of these cell lines did not result in the appearance of helper virus, indicating their stability. Additionally, the amphotropic nature of ψ CRIP-derived viruses was demonstrated by their ability to infect a wide range of mammalian cells. The study highlights the importance of developing safe and efficient packaging cell lines for in vivo gene transfer studies. The ψ CRIP and ψ CRE cell lines represent a significant advancement in this area, offering a reliable method for generating helper-free recombinant retroviruses with amphotropic and ecotropic host ranges. These cell lines are particularly valuable for studies involving cell lineage analysis and the development of human gene replacement therapies.Olivier Danos and Richard C. Mulligan developed packaging cell lines for generating helper-free recombinant retroviruses with amphotropic and ecotropic host ranges. These cell lines, named ψ CRIP and ψ CRE, were created by sequentially introducing two mutant Moloney murine leukemia virus (Mo-MuLV) proviral genomes with complementary mutations in the gag-pol or env regions into NIH 3T3 cells. Both genomes lacked the ψ sequence necessary for efficient viral particle encapsidation and had altered 3' ends. The resulting cell lines produced high-titer recombinant retroviruses without transferring packaging functions or generating helper virus, even under conditions where other cell lines could. This makes them valuable for in vivo gene transfer studies, particularly for cell lineage analysis and human gene replacement therapies. The initial step in generating recombinant retroviruses for mammalian gene transfer involves introducing a suitable proviral DNA vector into fibroblastic cells that produce viral proteins for encapsidation. Previous packaging cell lines, such as ψ-2 and ψ-AM, had issues with transferring packaging functions and generating helper virus. To address this, Danos and Mulligan introduced additional mutations into the ψ- genome to reduce the likelihood of these events. However, the possibility of recombinational events still existed, as mutations could be corrected by recombination with a ψ+ vector genome. To further eliminate the problem of packaging-function transfer and helper virus generation, Danos and Mulligan created two modified Mo-MuLV genomes with deletions of the ψ sequence and 3' LTR, encoding either the gag-pol or env gene. These genomes were introduced into NIH 3T3 cells through two rounds of transfection and coselection with different selectable markers. The resulting cell lines, ψ CRIP and ψ CRE, were characterized for their efficiency and safety. ψ CRIP produced amphotropic retroviruses, while ψ CRE produced ecotropic retroviruses. The packaging capacity of ψ CRIP and ψ CRE was tested using a mobilization assay, which showed that these cell lines did not transfer the ψ- genome or generate helper virus. Long-term cultivation of these cell lines did not result in the appearance of helper virus, indicating their stability. Additionally, the amphotropic nature of ψ CRIP-derived viruses was demonstrated by their ability to infect a wide range of mammalian cells. The study highlights the importance of developing safe and efficient packaging cell lines for in vivo gene transfer studies. The ψ CRIP and ψ CRE cell lines represent a significant advancement in this area, offering a reliable method for generating helper-free recombinant retroviruses with amphotropic and ecotropic host ranges. These cell lines are particularly valuable for studies involving cell lineage analysis and the development of human gene replacement therapies.