Single-Step Conversion of Cells to Retrovirus Vector Producers with Herpes Simplex Virus–Epstein-Barr Virus Hybrid Amplicons

Abstract

We report here on the development and characterization of a novel herpes simplex virus type 1 (HSV-1) amplicon-based vector system which takes advantage of the host range and retention properties of HSV–Epstein-Barr virus (EBV) hybrid amplicons to efficiently convert cells to retrovirus vector producer cells after single-step transduction. The retrovirus genes gag-pol andenv (GPE) and retroviral vector sequences were modified to minimize sequence overlap and cloned into an HSV-EBV hybrid amplicon. Retrovirus expression cassettes were used to generate the HSV-EBV-retrovirus hybrid vectors, HERE and HERA, which code for the ecotropic and the amphotropic envelopes, respectively. Retrovirus vector sequences encoding lacZwere cloned downstream from the GPE expression unit. Transfection of 293T/17 cells with amplicon plasmids yielded retrovirus titers between 106 and 107 transducing units/ml, while infection of the same cells with amplicon vectors generated maximum titers 1 order of magnitude lower. Retrovirus titers were dependent on the extent of transduction by amplicon vectors for the same cell line, but different cell lines displayed varying capacities to produce retrovirus vectors even at the same transduction efficiencies. Infection of human and dog primary gliomas with this system resulted in the production of retrovirus vectors for more than 1 week and the long-term retention and increase in transgene activity over time in these cell populations. Although the efficiency of this system still has to be determined in vivo, many applications are foreseeable for this approach to gene delivery.

Re-visiting the functional Relevance of the highly conserved Serine 40 Residue within HIV-1 p6Gag

“…A derivative of pNL4-3 unc FL in which the substitution at position 40 of p6 Gag was reversed to the wild-type Ser-codon while retaining all other substitutions (pNL4-3 unc FL-N40S) was also included (Figure 1 A). Mutant viruses were produced by transfection of HEK 293 T cells [ 13 ] using calcium phosphate and tested for efficiency of particle formation, Gag processing, Vpr incorporation, and infectivity. Controls included a release deficient late domain-defective variant (NL4-3 late(-), [ 14 ]), a derivative carrying alanine substitutions in the FRFG motif of p6 Gag and impaired in Vpr incorporation (NL4-3 Vpr(-), [ 3 ]), and a derivative which does not express Vpr (NL4-3 ΔVpr).…”

Identifying DNase I hypersensitive sites as driver distal regulatory elements in breast cancer

“…The HEK293T cell line 58 was chosen based on: (1) transfection has very high efficiency 59 ; (2) analysis of genes using BioGPS 60 within 500 kbp of the deleted putative driver DHSs showed that they are expressed in the cell line; (3) analysis of ChIP-seq data from HEK293T cells (Fig. 6b , Supplementary Fig.…”

A formylpeptide receptor, FPRL1, acts as an efficient coreceptor for primary isolates of human immunodeficiency virus

“…The human osteosarcoma cell line HOS [ 51 ] and CD4-transduced human glioma cell line U87/CD4 [ 52 , 53 ], as well as NP-2/CD4 [ 49 ], NP-2/CD4/CCR5 [ 49 ], NP-2/CD4/CXCR4 [ 49 ], and NP-2/CD4/FPRL1 (see below) cells were cultured in Eagle's minimum essential medium (NISSUI Co., Inc., Tokyo, Japan) supplemented with 10% FCS. The human embryonal kidney cell line 293T [ 54 ], human hepatoblastoma cell line HepG2 [ 55 ], human hepatoma cell line Huh7, and human astrocytoma cell line U251MG [ 56 ] were maintained in Dulbecco's modified Eagle minimum essential medium (NISSUI Co., Inc., Tokyo, Japan) supplemented with 10% FCS. Brain-derived fibroblast-like BT-20/N cells [ 53 , 57 ], derived from the surgically dissected human brain tissue of a patient with glioma and thought to originate from brain blood vessels, were cultured in RPMI 1640 medium containing 10% FCS, endothelial cell growth supplements (BD Bioscience, Medford, MA) (10 μg/ml), and epidermal growth factor (10 ng/ml).…”