Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system.
Cloning and sequencing of the cellular-viral junctions from the human adenovirus type 5 transformed 293 cell line. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. Graham F.L., Smiley J., Russell W.C., Nairn R. Large-scale manufacture and characterization of a lentiviral vector produced for clinical ex vivo gene therapy application. Merten O.W., Charrier S., Laroudie N., Fauchille S., Dugué C., Jenny C., Audit M., Zanta-Boussif M.A., Chautard H., Radrizzani M. Error bars represent means ± standard error of four independent experiments. Vector DNA copies (vector genomes /mL) in the lysate were determined by qPCR using primers for the CMV promoter sequence. The cells were collected 48 h later and freeze-thaw lysates were prepared. This was performed at small scale (B) and large scale (C). (B and C) AAV2 vectors were produced by transient transfection of a polyclonal 293T cell pool 27 using the pAAV2-NLS-GFP and pAAV2 RepCap plasmids, and the Ad helper plasmid 449B. Error bars represent means ± standard error of two or three independent experiments, and statistical analysis was performed using an unpaired student's T test. Functional titers were determined by FACS analysis. 293T and 293 refer to bulk HEK293T and HEK293 cells, respectively. Vector aliquots were titrated by transduction of HEK293 cells. The vector-containing supernatants were harvested at 72 h.
Sv40 poly a snapgene series#
Vector Production Using Knockout Clones Lentiviral vectors were produced by PEI-mediated transfection using a third-generation lentiviral vector system involving an EGFP-encoding vector plasmid. We have constructed a series of plasmids containing multiple polyadenylation signals downstream of the herpes simplex virus type 1 (HSV) thymidine kinase (tk)-coding region. The results obtained revealed that the lack of T antigen sequences did not impact AAV vector titers.ĪAV CRISPR/Cas9 HEK293T cells Lentivirus SV40 T antigen vector manufacturing. The capacity of the T antigen-negative cells to produce high titer adeno-associated virus (AAV) vectors was also evaluated. Lentiviral vectors produced using the T antigen null clones exhibited titers up to 1.5 × 10 7 transducing units (TU)/mL, while the titers obtained from the parent HEK293T cell line were up to 4 × 10 7 TU/mL. Western blot analysis of cell extracts prepared from the T antigen null clones confirmed that the SV40 large and small T antigen proteins were absent. The putative T antigen null clones demonstrated a loss of sequence reads mapping to T antigen-encoding sequences. Whole-genome (WG) and targeted locus amplification (TLA) sequencing of the parental HEK293T cell line revealed multiple SV40 T antigen-encoding sequences replacing cellular sequences on chromosome 3. Cell clones lacking T antigen-encoding sequences were identified using PCR. We used CRISPR-Cas9 genome editing to remove the SV40 T antigen-encoding sequences from HEK293T cells by transfecting them with a recombinant plasmid expressing Cas9 and two distinct single guide RNAs (sgRNAs) corresponding to the beginning and end of the T antigen coding region. Such cell-type and plasmid-construct dependency on gene expression from plasmids containing the SV40 DTS suggests that the gene expression from plasmids is not entirely dependent on its ability to enhance the nuclear import of said plasmids.The use of the human embryonic kidney (HEK) 293T cell line to manufacture vectors for in vivo applications raises safety concerns due to the presence of SV40 T antigen-encoding sequences. The gene expression from the plasmid constructs carrying the SV40 DTS varied with cell type and plasmid construct used. For this study, we used transient reporter gene expression assays on various cell types. In this report, we address the issue of the suitability of the SV40 DTS for cationic lipid-mediated gene delivery, and its capacity to improve the efficiency of the transfection process. 372-bp fragment of SV40 genomic DNA encompassing the SV40 promoter-enhancer-origin of replication (SV40 DTS), could enable the nuclear import of a plasmid carrying these sequences (Dean D.A. Recently, via microinjection into the cytoplasm and in situ hybridizations into a few cell types, it was shown that a region of Simian virus 40(SV40), specifically a c. One of the steps that limit transfection efficiency in non-viral gene delivery is inefficient nuclear import of plasmid DNA, once it has been delivered into the cytoplasm.
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