Transgenes delivered by lentiviral vector are suppressed in human embryonic stem cells in a promoter-dependent manner - PubMed (original) (raw)

Transgenes delivered by lentiviral vector are suppressed in human embryonic stem cells in a promoter-dependent manner

Xiaofeng Xia et al. Stem Cells Dev. 2007 Feb.

Abstract

Lentiviruses have been increasingly used for genetic modification of human cells including embryonic stem (ES) cells. Using four ubiquitous promoters--cytomegalovirus (CMV), cytomegalovirus immediate-early enhancer/chicken beta-actin hybrid (CAG), phosphoglycerate kinase (PGK), and human elongation factor-1alpha (EF1alpha)--in a lentiviral vector to drive the expression of the enhanced green fluorescent protein (EGFP) gene in human ES cells and mouse ES cells, we determined the extent of EGFP suppression by assessing the percentage of cells that were transduced with the EGFP gene but did not fluoresce green. A much higher level of transgene suppression was observed in human ES cells as compared to mouse ES cells. The suppression was also highly promoter dependent, leading to inactivation of more than 95% of the EGFP genes under the CMV or CAG promoter while only 55% under the PGK promoter. No promoter-dependent suppression was observed in transient transfection of human ES cells. Thus, the common phenomenon of poor transgene expression in human ES cells may be caused mainly by suppression of the transgene right after transduction and integration. Cautions should be taken to choose the optimal promoter when lentiviruses are used for genetic modification of human ES cells.

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Figures

FIG. 1

FIG. 1

EGFP expression in human H9 ES cells is promoter dependent. Human ES cells (the H9 line) were transduced with lentiviruses containing EGFP driven by different promoters at a MOI of 3 and analyzed 96 h later. (A) The bright-field and GFP channel views of representative transduced clones were shown in the left and middle columns, respectively. The promoters were indicated on the left. The numbers of fluorescent cells were then quantified by FACS and the representative results are shown in the right column. Two peaks were observed in FACS for each clone, with the left peak representing non-fluorescent cells while the right peak representing the fluorescent cell population. (B) The percentages of fluorescent cells were calculated from the FACS analyses. Results shown were the mean of three independent experiments with indicated standard deviations.

FIG. 2

FIG. 2

EGFP suppression in human H9 ES cells is promoter dependent. Genomic DNA of human ES cell clones transduced with various lentiviruses was extracted and used as template for PCR. (A) Regular PCR amplified a 300-bp fragment of the EGFP gene, showing that all the clones contain EGFP genes. The GAPDH gene was also amplified and used as a loading control. (B) The average copy numbers of the EGFP gene (± standard deviation) in the transduced clones were determined by qPCR using the human ACTB gene as an external control (n = 3). (C) The extent of suppression for each lentivector was calculated by dividing the percentage of nonfluorescent cells (results from Fig. 1B) by the average copy number of the EGFP gene. Results were plotted as the mean of three independent experiments with indicated standard deviations.

FIG. 3

FIG. 3

EGFP expression in mouse ES cells under different promoters. Mouse ES cells were transduced with different lentiviruses at MOI 3. (A) The bright-field and GFP channel views of representative clones 96 h after transduction were shown in the left and middle columns, respectively. The names of the promoters were indicated on the left. The numbers of fluorescent cells were then quantified by FACS and shown in the right column. (B) The percentages of fluorescent cells were quantified and plotted as the mean of three independent experiments with indicated standard deviations.

FIG. 4

FIG. 4

The degree of EGFP suppression in mouse ES cells is similar with different promoters. (A) All of the mouse ES clones transduced with different lentivirus were EGFP positive as shown by regular PCR using EGFP-specific primers. The GAPDH gene was amplified to show an equal amount of loading. (B) The average copy numbers of EGFP genes in the transduced clones were determined by qPCR using mouse ACTB gene as an external control (n = 3). (C) The extent of suppression for each lentivector was calculated by dividing the percentage of nonfluorescent cells (shown in Fig. 5B) by the average copy number of the EGFP gene. Results were plotted as the mean of three independent experiments with indicated standard deviations. A t_-test showed that the differences among CAG, PGK, and EF1_α promoters were not significant (p > 0.05).

FIG. 5

FIG. 5

Transient transfection efficiency in human H9 ES cells is independent of promoters. Human ES cells were transiently transfected with EGFP lentiviral transfer vectors containing EGFP driven by different promoters. (A) The brightfield and GFP channel views of representative transduced cells were shown in the left and right columns, respectively. The promoters were indicated on the left. (B) The numbers of fluorescent cells per mm2 of ES clone area were manually counted and plotted. Results shown are the mean of three independent experiments with indicated standard deviations. No significant difference between any two of the four promoters was detected by _t_-test (p > 0.05).

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