Stable packaging cell line producing pseudotyped retroviruses

摘要

The present invention relates to a stable, pseudotyped retrovirus packaging cell line comprising packaging cells which generate helper-free recombinant pseudotyped retrovirus. The packaging cell line comprises one or more non-retroviral expression constructs, such as an expression construct with the human cytomegalovirus (CMV) immediate early promoter or derivatives of this promoter (e.g., pMD), which direct expression of: (a) retroviral gagpol genes and (b) a non-retroviral gene which is under the control of an inducible operator system and whose gene product pseudotypes retroviral core virions. The present invention further relates to a method of making a stable, pseudotyped retrovirus packaging cell line which generates helper-free recombinant pseudotyped retrovirus. The present invention further relates to the particular packaging cell lines described herein (i.e., H29 gagpol, H29 new gagpol) and the particular cells and constructs (i.e., packaging elements) used to produce the stable, pseudotyped retrovirus packaging cell line described herein (e.g., H29 cells and pMD, pMDtet, pMDtet.G, PMD.gagpol, pMD.new gagpol constructs). The present invention relates to a retroviral vector for producing a cDNA library for expression in mammalian cells, comprising two retroviral long terminal repeats, a cloning site for insertion of cDNA and a cytomegalovirus promoter. The invention also relates to a cDNA library for expression in mammalian cells, the library comprising retroviral vectors of the present invention. The present invention also relates to a method of expression cloning in mammalian cells. The present invention also relates to a method of cDNA expression cloning in mammalian cells. The present invention also relates to a method of identifying a gene defect responsible for a mutant phenotype using cDNA expression cloning by complementation in mammalian cells.