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 EF1α 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 EF1α 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.
Bibliographical noteFunding Information:
We are grateful to Howard Cedar (The Hebrew University in Jerusalem, Israel) for providing the island element, Michel Revel (The Weizmann Institute, Rehovot, Israel) for the gift of foreskin fibroblasts, Nahum Sonenberg (McGill University, Montreal, ON, Canada) for providing poliovirus IRES, and Chaim Kahana (The Weizmann Institute, Rehovot, Israel) for the gift of Bip IRES. In addition we thank Michal Gropp from our lab for her professional support. This study was supported by grants from the Israeli Ministry of Science for The Strategic Center for Gene Therapy in Hadassah, NIH NINDS Grant RO1 NS046559-01, and the Horwitz and the Blum Foundations.
- dual promoter
- human embryonic stem cells
- lentiviral vector