1. ¹The Hadassah Human Embryonic Stem Cell Research Center, Hadassah University Medical Center, Jerusalem 91120, Israel
2. ²The Goldyne Savad Institute of Gene Therapy, Hadassah University Medical Center, Jerusalem 91120, Israel
3. ³Department of Gynecology, Hadassah University Medical Center, Jerusalem 91120, Israel

Correspondence: Benjamin E. Reubinoff, The Hadassah Human Embryonic Stem Cell Research Center, The Goldyne Savad Institute of Gene Therapy, Hadassah University Hospital, P.O. Box 12000, Ein-Kerem, Jerusalem 91120, Israel. Fax: +972 2 6430982. E-mail: reubinof@md.huji.ac.il

Received 30 June 2005; Revised 9 February 2006; Accepted 12 February 2006.

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 EF1 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 EF1 promoter. In summary, we developed bicistronic and novel dual-promoter LVs that enable high and homogeneous expression of transgenes by polyclonal hESCs after antibiotic selection. These vectors may provide important tools for basic and applied research on hESCs.