No abstract

Retrovirus vectors can be made in the absence of helper virus by using retrovirus packaging cell lines. Helper-free virus is critical for a variety of gene transfer studies. The most useful packaging cell lines contain helper virus DNA from which the signal required for packaging of the viral RNA genome into virions has been deleted. However, we showed that the ability to package virus is conferred at very low frequency to cells infected with virus from these packaging cell lines, presumably by low-frequency transmission of the deleted virus genome. In addition, these packaging cell lines can interact with some retroviral vectors to yield replication-competent virus. We constructed packaging cell lines containing helper virus DNA that had several alterations in addition to deletion of the packaging signal. The new packaging cells retained the useful features of previously available lines but did not yield helper virus after introduction of any of the vectors tested, and transfer of the packaging function was not detected.

MyoD is a master regulatory gene for myogenesis. Under the control of a retroviral long terminal repeat, MyoD was expressed in a variety of differentiated cell types by using either a DNA transfection vector or a retrovirus. Expression of muscle-specific proteins was observed in chicken, human, and rat primary fibroblasts and in differentiated melanoma, neuroblastoma, liver, and adipocyte lines. The ability of MyoD to activate muscle genes in a variety of differentiated cell lines suggests that no additional tissuespecific factors other than MyoD are needed to activate the downstream program for terminal muscle differentiation or that, if such factors exist, they are themselves activated by MyoD expression.

Cell surface receptors for gibbon ape leukemia virus (Glvr-1) and marine amphotropic retrovirus (Ram-i) Ram-i (10) and human Glvr-1 (7) cDNAs were cloned into pGEM-7Z (Promega) with their 5' ends adjacent to the SP6 promoter. For mRNA synthesis, the plasmids were linearized and transcribed with SP6 polymerase in the presence of m7G(5')ppp(5')G caps according to the manufacturer's directions (Pharmacia). Xenopus laevis oocytes were injected with 50 nl of mRNA (1 ng/nl) or with an equal volume of H20 and were incubated for 4-6 days at 17'C; then two-microelectrode voltage-clamp recordings or radiolabel uptake assays were performed at room temperature as described (23).Abbreviations: GALV, gibbon ape leukemia virus; Glvr-1, cell surface receptor for GALV; Ram-i, cell surface receptor for amphotropic murine retrovirus; HIV, human immunodeficiency virus; MLV, murine leukemia virus; Mo-MLV, Moloney MLV.