Enhancement of Transgene Expression by HDAC Inhibitors in Mouse Embryonic Stem Cells

Abstract

Embryonic stem (ES) cells can self-renew and differentiate to various cells depending on the culture condition. Although ES cells are a good model for cell type specification and can be useful for application in clinics in the future, studies on ES cells have many experimental restraints including low transfection efficiency and transgene expression. Here, we observed that transgene expression after transfection was enhanced by treatment with histone deacetylse (HDAC) inhibitors such as trichostatin A, sodium butyrate, and valproic acid. Transfection was performed using conventional transfection reagents with a retroviral vector encoding GFP under the control of CMV promoter as a reporter. Treatment of ES cells with HDAC inhibitors after transfection increased population of GFP positive cells up to 180% compared with untreated control. ES cells showed normal expression of stem cell markers after treatment with HDAC inhibitors. Transgene expression was further enhanced by modifying transfection procedure. GFP positive cells selected after transfection were proved to have the stem cell properties. Our improved protocol for enhanced gene delivery and expression in mouse ES cells without hampering ES cell properties will be useful for study and application of ES cells.

Keywords: Enhancement; HDAC inhibitor; Mouse embryonic stem cell; Optimization.; Transfection.

Fig. 1

Increase of transgene expression by HDAC inhibitors in mouse ES cells. Population of GFP positive cells was assessed by FACS analysis at 24 hr after transfection with Lipofectamine™ 2000 (A) and FuGENE^® HD (B). Transfected ES cells were treated with HDAC inhibitors at the indicated concentration for the incubation period. Relative percentage of GFP positive cells compared to the control is shown graphically taking the control as 100%. The experiments were performed at least three times. *P<0.05, **P<0.01 by paired student t-test.

Fig. 2

Optimization of condition for effective transfection using HADC inhibitors. Population of GFP positive cells was determined by FACS analysis at 24 hr after transfection with Lipofectamine™ 2000. Transfected ES cells were treated with HDAC inhibitors such as NaB (A), VPA (B) and TSA (C) at the indicated concentration for the incubation period. The percentage of GFP positive cells was monitored varying the plating time of mouse ES cells before transfection (6 hr, 9 hr, and 12 hr).

Fig. 3

Characterization of transfected mouse ES cells. GPF positive mouse ES cells were characterized after transfection according to the optimized protocol. (A) Expression of stem cell markers was determined by RT-PCR. Expression of Oct4 and Nanog mRNA was analyzed. (B) Expression of stem cells markers such as Oct4, Sox-2, Klf-4, and Nanog was determined by FACS analysis after immunostaining with respective antibodies.

Fig. 4

Selection of G418 resistant cells and characterization of the transgenic ES cells. (A) Mouse ES cells were selected with G418 after transfection with pMSCV-GFP using Lipofectamine™ 2000 and NaB; high percentage of cells expressed GFP. (B) Stem cell marker expression in GFP positive mouse ES cells was determined by FACS analysis.