Plat-E: an efficient and stable system for transient packaging of retroviruses

Abstract

A potent retrovirus packaging cell line named Platinum-E (Plat-E) was generated based on the 293T cell line. Plat-E is superior to existing packaging cell lines regarding efficiency, stability and safety. The novel packaging constructs utilized in establishment of Plat-E ensure high and stable expression of viral structural proteins. Conventional packaging constructs made use of the promoter of MuLV-LTR for expression of viral structural genes gag-pol and env, while our packaging constructs utilized the EF1␣ promoter, which is 100-fold more potent than the MuLV-LTR in 293T cells in combination with the Kozak's consensus sequence upstream of the initiation codon resulting in high expression Keywords: packaging cell; retroviruses; ecotropic; EF1␣ promoter Retroviral vectors and packaging cells are important tools for gene transfer applications. Introduction of retroviral vectors containing the gene of interest into suitable packaging cells enables production of infectious retroviruses, and these particles can infect target cells and stably transmit the gene of interest into chromosomes. In conventional strategies, stable high producers of a retrovirus vector harboring a gene of interest were established by transducing the retrovirus construct into NIH3T3-based packaging cells such as PA317, 1 and 2-3 months were usually needed to acquire high producers. Pear et al 2 developed a unique packaging system by which high titer retroviruses can be obtained in 3 days by transient transfection. The expression of viral structural genes was driven by the MuLV LTR in Bosc23 cells. For transient transfection, the combination of Bosc23 cells and the pMX-neo vector 3 produced 1-3 × 10 6 /ml viruses, assessed based on the number of neomycin resistant colonies of the infected NIH3T3 cells (data not shown). Since Bosc23 cells carry the large T antigen, we attempted to increase titers of the retroviruses by introducing the SV40 origin to the pMX vector for amplification of the vector. However, this proved unfeasible (data not shown), suggesting that the limiting factor was the expression level of the viral structural proteins in the packaging cells. of virus structural proteins in Plat-E cells. To maintain the high titers of retroviruses under drug selection pressure, we inserted the IRES (internal ribosome entry site) sequence between the gene encoding gag-pol or env, and the gene encoding a selectable marker in the packaging constructs.Plat-E cells can stably produce retroviruses with an average titer of 1 × 10 7 /ml for at least 4 months. In addition, as we used only the coding sequences of viral structural genes to avoid inclusion of unnecessary retrovirus sequences in the packaging constructs, the probability of generating the replication competent retroviruses (RCR) by recombination can virtually be ruled out. Gene Therapy (2000) 7, 1063-1066. together with the plasmid encoding another selection gene GPT (guanine phosphoribosyl transferase), one after the other. Therefore, expression of selectable markers did not guarantee ...

Mechanisms controlling titer and expression of bidirectional lentiviral and gammaretroviral vectors

“…GV cell-free supernatants were generated by transient transfection of 293T cells seeded in 10 cm cell culture dishes, using plasmids encoding the viral vector, gag/ pol and an ecotropic 58 or VSV-G 59 envelope proteins. LV supernatants were produced accordingly by cotransfection of LV vector, gag/pol, envelope and a Rev encoding plasmid.…”

A role for Myc in facilitating transcription activation by E2F1

“…Human glioblastoma T98G cells and Plat-A cells (Morita et al, 2000) were maintained in Dulbelco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). T98G cells were synchronized in G 0 phase by starvation in DMEM supplemented with 0.1% FBS for 48 h and then stimulated to re-enter the cell cycle by addition of FBS to a final concentration of 20%.…”

“…For ChIP assays to determine histone acetylation as a function of Myc, retroviruses expressing shRNAs were utilized to target Myc expression by cloning shRNA sequences into the pSuperior vector (Oligoengine). Retroviruses were packaged using Plat-A cells for infection of T98G cells (Morita et al, 2000). Plasmid DNA was transfected into the packaging line using Fugene 6 transfection reagent (Roche, Basel, Switzerland).…”

Growth control of hybridoma cells with an artificially induced EpoR-gp130 heterodimer

“…A retroviral packaging cell line, Plat-E (Morita et al 2000), was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 1 lg/ml puromycin (Sigma, St. Louis, MO) and 10 lg/ml blasticidin (Kaken Pharmaceutical, Tokyo, Japan).…”