A method for producing high-titer, helper-free retroviruses using transient transfection of the BOSC 23 cell line is described. The BOSC 23 cell line, derived from the Ad5-transformed 293 cell line, is highly transfectable and can produce infectious retroviruses at titers exceeding 10^7 infectious particles per milliliter within 72 hours after transfection. This system allows for the production of high-titer retroviral vectors expressing genes that are difficult to propagate in stable producer lines. The method is useful for gene therapy and the efficient transfer of recombinant genetic material into rare cells, such as stem cells and neurons. The BOSC 23 system can produce high-titer retroviruses without the need for selection, and it is free of replication-competent virus. This was demonstrated by a stringent assay in which the presence of helper virus would rescue the integrated lacZ-containing provirus from BAG cells. No rescue was observed using three different retroviral constructs, including one with an extended retroviral RNA packaging site. The BOSC 23 system is also capable of producing retroviruses that can infect early hematopoietic progenitors. This was shown by testing the ability of BOSC 23 cells transfected with a retroviral vector to infect hematopoietic progenitors in a colony-forming unit-spleen assay. The BOSC 23 system has several advantages, including the ability to produce high-titer retroviruses expressing genes that are detrimental to the growth of stable producer cell lines. This is because BOSC 23 cells are exposed to the retroviral products for less than 72 hours, minimizing the detrimental effects of the gene product on the producer cell line. The BOSC 23 system is also free of detectable replication-competent virus. This was demonstrated by the use of a stringent assay in which either packaging of the helper functions or production of helper virus itself would result in the rescue of the integrated lacZ-containing provirus from the BAG cells. No rescue was seen using three different retroviral constructs, including one with an extended retroviral RNA packaging site. The BOSC 23 system has many potential applications, including testing the efficacy of different retroviral vectors and conditions, overexpressing proteins, and creating animal models of disease. The present work also suggests that an amphotropic counterpart to the BOSC 23 cell line may be useful in human gene therapy.A method for producing high-titer, helper-free retroviruses using transient transfection of the BOSC 23 cell line is described. The BOSC 23 cell line, derived from the Ad5-transformed 293 cell line, is highly transfectable and can produce infectious retroviruses at titers exceeding 10^7 infectious particles per milliliter within 72 hours after transfection. This system allows for the production of high-titer retroviral vectors expressing genes that are difficult to propagate in stable producer lines. The method is useful for gene therapy and the efficient transfer of recombinant genetic material into rare cells, such as stem cells and neurons. The BOSC 23 system can produce high-titer retroviruses without the need for selection, and it is free of replication-competent virus. This was demonstrated by a stringent assay in which the presence of helper virus would rescue the integrated lacZ-containing provirus from BAG cells. No rescue was observed using three different retroviral constructs, including one with an extended retroviral RNA packaging site. The BOSC 23 system is also capable of producing retroviruses that can infect early hematopoietic progenitors. This was shown by testing the ability of BOSC 23 cells transfected with a retroviral vector to infect hematopoietic progenitors in a colony-forming unit-spleen assay. The BOSC 23 system has several advantages, including the ability to produce high-titer retroviruses expressing genes that are detrimental to the growth of stable producer cell lines. This is because BOSC 23 cells are exposed to the retroviral products for less than 72 hours, minimizing the detrimental effects of the gene product on the producer cell line. The BOSC 23 system is also free of detectable replication-competent virus. This was demonstrated by the use of a stringent assay in which either packaging of the helper functions or production of helper virus itself would result in the rescue of the integrated lacZ-containing provirus from the BAG cells. No rescue was seen using three different retroviral constructs, including one with an extended retroviral RNA packaging site. The BOSC 23 system has many potential applications, including testing the efficacy of different retroviral vectors and conditions, overexpressing proteins, and creating animal models of disease. The present work also suggests that an amphotropic counterpart to the BOSC 23 cell line may be useful in human gene therapy.