Integration-deficient lentivectors: an effective strategy to purify and differentiate human embryonic stem cell-derived hepatic progenitors
- Equal contributors
1 INSERM U 972, IFR 93, Bicêtre Hospital, and Paul Brousse Hospital, Villejuif F-94807, France
2 Université Paris-Sud, UMR-S972, IFR 93, Bicêtre Hospital and Paul Brousse Hospital, Villejuif F-94807, France
3 Vectalys SAS, Canal Biotech 2, 3 rue des satellites, Toulouse F-31400, France
4 CIBER de Diabetes y Obesidad, Centro de Investigación Principe Felipe, Eduardo Primo Yúfera 3, Valencia 46012, Spain
5 Department of Cytogenetics, Béclère Hospital, Clamart F-92141, France
6 Department of Surgery, The Anne McLaren Laboratory for Regenerative Medicine, Cambridge CB2 0SZ, UK
BMC Biology 2013, 11:86 doi:10.1186/1741-7007-11-86Published: 19 July 2013
Additional file 1: Figure S1:
Transient green fluorescent protein (GFP) expression after transduction of human embryonic stem cells (hESCs) with elongation factor (EF)1α-GFP integrase-defective lentivectors (IDLVs). (A) Time course of fluorescence-activated cell sorting (FACS) analysis used to analyze fractions of fluorescent GFP-expressing cells after transduction of H9 cells with EF1α-GFP-IDLV at a multiplicity of infection (MOI) of 10 and 30. (B) Time course of FACS analysis showing similar proportions of GFP-IDLV cells and GFP-integrating lentivectors (ILV) cells 3 days after transduction, and a decrease in the proportion of GFP-IDLV cells 10 and 14 days after transduction, and of EF1α-GFP-ILV cells in the presence of raltegravir.
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Additional file 2: Figure S2:
(A) Genetic map of 1-long terminal repeat (LTR) and 2-LTR circular DNA. (B) Map of the probe used for the Southern blotting experiments. After double BamHI/EcoRI digestion, a 1,316 bp band common to integrated and non-integrated forms of the lentivector DNA was detected. After single EcoRI digestion, only non-integrated forms (1-LTR and 2-LTR circle) of the lentivector DNA were detected.
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Additional file 3: Figure S3:
Limits of detection of virus copy number. Serial dilutions (1:50 to 1:10000) of genomic DNA from a clonal cell line containing one copy number of an integrating green fluorescent protein (GFP)-expressing lentivirus (D2) and derived from HCT 116 cekks. HCT 116 NT: control non-transduced cells.
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