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1.
Figure 3

Figure 3. Overexpression of Sall4 enhanced Oct4-gfp expression and ES cell-like colony formation in MEFs during reprogramming.. From: High-Efficiency Stem Cell Fusion-Mediated Assay Reveals Sall4 as an Enhancer of Reprogramming.

The percentage of GFP positive MEFs at: A) 24 h and B) 48 h post-fusion. C) The number of GFP positive, G418-resistant colonies in 1 well of a 6-well plate, 10 days post-fusion. The overexpression of Sall4 positively enhanced both Oct4-gfp expression and colony formation of MEFs upon reprogramming.

Connie C. Wong, et al. PLoS ONE. 2008;3(4):e1955.
2.
Figure 5

Figure 5. The N-terminal domain is not required for Sall4 function in reprogramming.. From: High-Efficiency Stem Cell Fusion-Mediated Assay Reveals Sall4 as an Enhancer of Reprogramming.

A) Sall4 d12 mutant behaved similarly to wt Sall4 in reprogramming. Sall4 d12, as well as two clones of wt Sall4 of identical sequences, were overexpressed in Oct4-gfp MEFs, which were then fused to ESCs and assayed for GFP expression as described. B) The overexpression of Sall4 d12 resulted in a similar increase in the number of Oct4-gfp cells as wt Sall4, as well as a similar increase in the number of reprogrammed colonies.

Connie C. Wong, et al. PLoS ONE. 2008;3(4):e1955.
3.
Figure 2

Figure 2. Screen of positive regulators of somatic cell reprogramming.. From: High-Efficiency Stem Cell Fusion-Mediated Assay Reveals Sall4 as an Enhancer of Reprogramming.

A) Schematic of the screen. Candidate genes were overexpressed in Oct4-gfp, G418-resistant MEFs via lentivirus infection 72 h before fusion. B) Successful lentiviral overexpression was verified by Western blotting, as well as expression of the positive control mCherry. C) Infected MEFs were harvested at 24 h before fusion and stained with the fluorescent dye Vybrant DiD. Prepared MEFs were plated with unstained ESCs. GFP expression was FACS analyzed at 24 and 48 h post-fusion. G418 was added to the fused cells at 48 h post-fusion, and the formation of G418-resistant, GFP positive colonies was assayed 10 days post-fusion. D) The visible overexpression of mCherry in infected MEFs indicated the effectiveness of our lentiviral overexpression system. Scale bar represents 50 ┬Ám.

Connie C. Wong, et al. PLoS ONE. 2008;3(4):e1955.
4.
Figure 4

Figure 4. Sall4 is a bona fide positive regulator of reprogramming.. From: High-Efficiency Stem Cell Fusion-Mediated Assay Reveals Sall4 as an Enhancer of Reprogramming.

A) Overexpression of Sall4 in Oct4-gfp MEFs did not induce GFP expression. Sall4, mCherry and luciferase were overexpressed in Oct4-gfp MEFs via lentivirus infection. Half of the infected MEFs was fused to ESCs as described, while the other half was not. Only MEFs overexpressing Sall4 and fused to ESCs showed an increased number of GFP positive cells when compared to the negative controls, indicating that overexpression of Sall4 alone did not induce GFP expression. The numbers of GFP positive cells in the infected cells relative to that of the uninfected cells were shown. B) Overexpression of Sall4 did not increase cell doubling time in MEFs. mCherry and two different constructs of Sall4 were overexpressed in MEFs, which were plated onto 6 well plates and assayed for cell number every 24 h. Note that another clone of Sall4 of identical sequence was used as a duplicate. C, D) Infection of Sall4-expressing lentiviruses did not increase the colony formation efficiency in ESCs. Both ESCs and previously reprogrammed MEFs that were tetraploid were infected with lentiviruses expressing Sall4, mCherry or luciferase. D) The infected cells were plated either on gelatin or on feeder cells. The number of colonies formed was assayed after 7 days.

Connie C. Wong, et al. PLoS ONE. 2008;3(4):e1955.
5.
Figure 1

Figure 1. Establishment of an efficient fusion assay.. From: High-Efficiency Stem Cell Fusion-Mediated Assay Reveals Sall4 as an Enhancer of Reprogramming.

A) Fusion efficiency of the assay. The MEFs and ESCs were stained with the fluorescent dyes Vybrant DiD and Vybrant DiO respectively before fusion. Fusion efficiency was determined by FACS analysis at 5 h post-fusion, using an unfused mixture of cells as a negative control. Note that previous studies have shown that cell surface dyes rarely diffuse across the cell membranes of stained cells [41]. The dual-labeled cells in the unfused population was most likely due to non-specific binding between the ESCs and MEFs. The green fluorescent dye Vybrant DiO was only used in the determination of fusion efficiency but not in a typical reprogramming experiment (due to interference with the observation of GFP signal). B) Morphology change of MEFs during reprogramming. The forward- and side-scatter profiles of the GFP positive cells were FACS analyzed at 24, 48 and 72 h post-fusion. The morphology of the reprogrammed MEFs changed with time to resemble that of the ESCs. C) Quantification of GFP expression. The number of GFP positive cells was FACS analyzed at 24 and 48 h post-fusion (right panel), using wildtype MEFs that did not carry the Oct4-gfp transgene but had undergone identical fusion treatment with ESCs as a negative control (left panel). The number of GFP positive cells from wt MEFs was subtracted from that of Oct4-gfp MEFs in all calculations.

Connie C. Wong, et al. PLoS ONE. 2008;3(4):e1955.

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