Lentiviral vectors harboring a dual-gene system allow high and homogeneous transgene expression in selected polyclonal human embryonic stem cells

Molecular Therapy : the Journal of the American Society of Gene Therapy
Israel Ben-DorB E Reubinoff

Abstract

Genetic modification of human embryonic stem cells (hESCs) is highly valuable for their exploitation in basic science and therapeutic applications. Here we developed lentiviral vectors (LVs) constitutively expressing a reporter and a selectable marker to enable high and homogeneous transgene expression within polyclonal hESCs. LVs carrying GFP and a downstream puromycin resistance gene, linked by the encephalomyocarditis virus (EMCV) or poliovirus internal ribosome entry sites (IRES), allowed homogeneous GFP expression after antibiotic selection. The GFP-expression levels were higher with the EMCV IRES. We also developed dual-promoter vectors harboring a reporter and an antibiotic resistance gene under the regulation of human EF1alpha and PGK1 promoters, respectively. Optimal efficiency was obtained when: (1) the reporter cassette was upstream rather than downstream of the selectable marker cassette, (2) the puromycin rather than the neomycin resistance gene was used, (3) a 5' deletion (314 bp) was created in the PGK promoter, and (4) two copies of a 120-bp element derived from the hamster Aprt CpG island were introduced upstream of the EF1alpha promoter. In summary, we developed bicistronic and novel dual-promoter LVs that ena...Continue Reading

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Citations

Jul 5, 2007·Molecular Therapy : the Journal of the American Society of Gene Therapy·Sunghoi HongKwang-Soo Kim
Oct 3, 2009·Tissue Engineering. Part a·Sujoy K DharaSteven L Stice
May 31, 2011·The Journal of Biological Chemistry·Kamyar HadianDaniel Krappmann
Mar 2, 2013·Human Gene Therapy Methods·Naoya UchidaTakashi Shimada
Mar 14, 2007·Stem Cells and Development·Cory R NicholasAndrew D Leavitt
Sep 6, 2007·Stem Cells and Development·Rishi Sharma, Ram Raghubir
Aug 24, 2010·Cellular Reprogramming·Katsuhisa TashiroHiroyuki Mizuguchi
Jan 9, 2007·Transfusion·Yoriko Saito
Oct 23, 2014·Stem Cells·Etti Ben-ShushanBenjamin E Reubinoff
May 26, 2011·Neuron·Steve S W HanKevin C Eggan
Jul 12, 2007·Molecular Therapy : the Journal of the American Society of Gene Therapy·Helen GharwanDavid W Russell
Mar 27, 2010·Biomedical and Environmental Sciences : BES·Chen-Yu XuNing-Shao Xia
Sep 21, 2012·World Journal of Stem Cells·Dmitry A OvchinnikovErnst J Wolvetang
Jun 4, 2010·Lab on a Chip·Jun TianStelios T Andreadis
Jul 31, 2013·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Yaa-Jyuhn James MeirSareina Chiung-Yuan Wu
Jan 22, 2019·International Journal of Cell Biology·Masoumeh RostamiMajid Shahbazi
Dec 20, 2019·Journal of the International AIDS Society·Youry KimJenny L Anderson
Dec 4, 2012·Plant Science : an International Journal of Experimental Plant Biology·Young Sim SonJeong Dong Bahk

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