The authors have developed a set of packaging cell lines for generating helper-free recombinant retroviruses with amphotropic and ecotropic host ranges. To address the issues of packaging function transfer and helper virus formation, two mutant Moloney murine leukemia virus (Mo-MuLV) proviral genomes carrying complementary mutations in the gag-pol or env regions were introduced into NIH 3T3 cells by cotransfection. These genomes contained a deletion of the ψ sequence necessary for efficient encapsidation and additional alterations at the 3' end. The resulting packaging cell lines, ψCRIP and ψCRE, can produce high titers (10^8 colony-forming units/ml) of recombinant retroviruses with amphotropic and ecotropic host ranges, respectively. Importantly, viral producers derived from these packaging cell lines do not transfer packaging functions or yield helper virus, even under conditions where existing packaging cell lines can transfer packaging functions and/or generate helper virus. These properties make the ψCRIP and ψCRE packaging lines valuable for in vivo gene transfer studies aimed at cell lineage analysis and human gene replacement therapies. The study also highlights the importance of preventing the transfer of packaging functions and the formation of helper viruses to ensure safety and efficiency in gene transfer applications.The authors have developed a set of packaging cell lines for generating helper-free recombinant retroviruses with amphotropic and ecotropic host ranges. To address the issues of packaging function transfer and helper virus formation, two mutant Moloney murine leukemia virus (Mo-MuLV) proviral genomes carrying complementary mutations in the gag-pol or env regions were introduced into NIH 3T3 cells by cotransfection. These genomes contained a deletion of the ψ sequence necessary for efficient encapsidation and additional alterations at the 3' end. The resulting packaging cell lines, ψCRIP and ψCRE, can produce high titers (10^8 colony-forming units/ml) of recombinant retroviruses with amphotropic and ecotropic host ranges, respectively. Importantly, viral producers derived from these packaging cell lines do not transfer packaging functions or yield helper virus, even under conditions where existing packaging cell lines can transfer packaging functions and/or generate helper virus. These properties make the ψCRIP and ψCRE packaging lines valuable for in vivo gene transfer studies aimed at cell lineage analysis and human gene replacement therapies. The study also highlights the importance of preventing the transfer of packaging functions and the formation of helper viruses to ensure safety and efficiency in gene transfer applications.