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1.
FIGURE 8

FIGURE 8. From: High-level Genomic Integration, Epigenetic Changes, and Expression of Sleeping Beauty Transgene.

Effect of SB-IHK-DsRed insertion on the expression of adjacent endogenous host genes. (A) Total RNA purified from K562 control cells was used as template for RT-PCR with primer sets correspondent to RGS13, XM_001128367.1, PRKGQ, SNCAIP and XP_001717916.1, respectively. The RT-PCR products were subjected to agarose gel electrophoresis. (B) Total RNA was purified from cell clone 8, 16, MS39 and 41, respectively, and used as template for RT-PCR of the closest downstream host genes: RGS13 for clone 8; XM_001128367.1 and PRKGQ for clone 16; SNCAIP for clone MS39; and XP_001717916.1 for clone 41. XM_001128367.1 and XP_001717916.1 are abbreviated as xm112 and xp171, respectively. (C) The control RT-PCR of GAPDH was carried out to verify equal amount of template for each sample.

Jianhui Zhu, et al. Biochemistry. ;49(7):1507.
2.
FIGURE 2

FIGURE 2. From: High-level Genomic Integration, Epigenetic Changes, and Expression of Sleeping Beauty Transgene.

Genomic copy number and chromosomal location of SB-IHK-DsRed insertions in individual K562 clones. (A) Genomic DNA isolated from single-cell clones was used as template for PCR using primers specific for DsRed and for the endogenous genomic HS3. (B) Determination of the SB-Tn insertion sites in cell clones. Using ligation-mediated nested PCR of the isolated genomic DNA, the insertion sites of the SB-IHK-DsRed were amplified and sequenced. NCBI BLAST analysis of the recovered flanking sequences was used to determine the genomic insertion sites in the K562 clones. The proportion of DsRed+ cells in each individual clones was determined by FACS analysis. Number of insertion loci identified by ligation-mediated PCR corresponded with the number estimated by semi-quantitative PCR in panel (A). MS prefix represents cell clones isolated by FACS machine whereas other cell clones were isolated manually.

Jianhui Zhu, et al. Biochemistry. ;49(7):1507.
3.
FIGURE 6

FIGURE 6. From: High-level Genomic Integration, Epigenetic Changes, and Expression of Sleeping Beauty Transgene.

Increased DsRed expression following treatments to reverse epigenetic modifications of genomic DNA. FACS analyses of clones 51-11 and 51-9 following treatment with 5-Aza-2′-deoxycytidine (5-aza) to inhibit DNA methylation (A, left panel) or with Trichostatin A (TSA) to block histone deacetylation of the chromatin (A, right panel) showed the increase of DsRed expression. The data (mean ± 1 SD) represent results from three independently treated cultures with each reagent. The clones and the number of treatment days with each chemical reagent are indicated below the horizontal axis. (B) Representative FACS analyses are shown that were performed after treatment for 6 days with either 5-aza or TSA, and the percentage of DsRed+ cells is indicated in the upper left of each plot.

Jianhui Zhu, et al. Biochemistry. ;49(7):1507.
4.
FIGURE 4

FIGURE 4. From: High-level Genomic Integration, Epigenetic Changes, and Expression of Sleeping Beauty Transgene.

IHK promoter is repressed following megakaryocyte differentiation. Clones 14, 18, 49 and two other clones, 1 and 23 were treated with PMA to induce megakaryocytic differentiation. (A) The clones were stained for CD235a (FITC) and CD41 (APC) as an erythroid-specific and megakaryocytic marker, respectively, and analyzed by flow cytometry against DsRed (vertical axis) and CD41 expression (horizontal axis) before (upper panel) and after PMA induction (lower panel). (B) Flow cytometry plots of CD235a+ (vertical axis) versus CD41+ (horizontal axis) cells on the same set of cell clones shown in (A). Upper panels represent flow cytometry of cell clones without PMA induction, and lower panels are flow cytometry of the cell clones after PMA induction. (C) Plots of CD235a signals of CD41+ and DsRed+ cells in clone 49 (panel A, lower right end) versus that of the parental K562 isotype control. The signal of dual-positive clone 49 is shown in red, the isotype control of K562 is shown in blue.

Jianhui Zhu, et al. Biochemistry. ;49(7):1507.
5.
FIGURE 3

FIGURE 3. From: High-level Genomic Integration, Epigenetic Changes, and Expression of Sleeping Beauty Transgene.

The IHK promoter directs erythroid-specific expression of DsRed. SB-IHK-DsRed single clones, representing low, medium and high levels of cells expressing DsRed were used to track the modulation of DsRed expression, following treatment to induce erythroid differentiation. (A) Fluorescent microscopy of the clones 14, 49, MS27 and MS28 showing the DsRed expression prior to treatment. (B) Flow cytometry of the cell clones with DsRed (vertical axis) and erythroid specific marker, CD235a, (horizontal axis), under normal culture condition (upper panels) or under hemin treatment for 3 days to induce erythroid differentiation (lower panels). At far left is the parental K562 cells stained with mouse isotype antibody-FITC as a negative control, while the individual clones are identified above the FACS panels. (C) The western blot analysis of the cell clones against SB10, γ-globin, and β-actin control, respectively. The clone ID number as well as presence or absence of hemin treatment are indicated above the lanes.

Jianhui Zhu, et al. Biochemistry. ;49(7):1507.
6.
FIGURE 7

FIGURE 7. From: High-level Genomic Integration, Epigenetic Changes, and Expression of Sleeping Beauty Transgene.

DNA methylation at the inserted SB-Tns and endogenous host copy of ANKYRIN1 promoter. (A) COBRA analyses to establish overall CpG methylation levels at the investigated regions. Bisulfite PCR products digested with TaqI restriction endonuclease (T) were compared with uncut controls (N) for each samples of different SB clones. (B) Methylation at each CpG dinucleotide in the 620 bp region indicated under the schematic map was profiled by bisulfite-mediated genomic sequencing. Open circles represent unmethylated CpGs while filled circles are methylated CpGs. Each horizontal line of circles is derived from a single bisulfite PCR amplicon. Bisulfite-PCR amplicons derived from a cell clone were organized into each block with the clone number indicated at right. The end of the ANKYRIN1 promoter sequence and start of the DsRed CDS are indicated by the solid line through the blocks, with the above arrow indicating the direction of transcription. (C) CpG methylation profile at the endogenous host copy of ANKYRIN1 promoter. The region identical with ANKYRIN1 promoter sequence in the SB-IHK-DsRed is indicated in the schematic representation above the blocks of CpG methylation data derived from individual bisulfite-PCR amplicons. The cell clone from which genomic DNA was isolated is indicated at right, and methylated CpGs indicated by filled circles. The absence of CpG methylation at this region was also verified in the cell clone MS14 (data not shown).

Jianhui Zhu, et al. Biochemistry. ;49(7):1507.
7.
FIGURE 5

FIGURE 5. From: High-level Genomic Integration, Epigenetic Changes, and Expression of Sleeping Beauty Transgene.

Long-term expression of DsRed in individual clones. (A) Flow cytometry analysis of DsRed expression in clones at 1 month (upper panel) and at 6 months (lower panel). The clone number is indicated above and the percent DsRed+ cells indicated in the upper left corner of each plot. (B) Analysis of DsRed expression by flow cytometry in two individual subclones 51-9 (65.7% DsRed+) and 51-11 (4.0% DsRed+), both of which were derived at 6 months from the same DsRed-positive cell clone 51 which had dropped from 74% of the cells expressing DsRed at 1 month to 5% at 6 months. Clones were stained for the erythroid specific marker CD235a, and the percentage of single or dual positive cells was determined by FACS analysis. The percentages of the population positive or negative for DsRed (vertical axis), and/or CD235a (horizontal axis) are indicated in the upper or lower corner of the relevant quadrant of the FACS plot. (C) Ligation-mediated PCR analysis of the genomic insertion sites in the parental clone 51 and the two subclones 51-9 and 51-11. Clone 51, sub-clones 51-9, and 51-11 gave the same product bands, a and b, which when sequenced were determined to be the same flanking host genomic sequences as those flanking the dual insertion sites in the parental clone 51 (Figure 2A). M, DNA ladder.

Jianhui Zhu, et al. Biochemistry. ;49(7):1507.
8.
FIGURE 1

FIGURE 1. From: High-level Genomic Integration, Epigenetic Changes, and Expression of Sleeping Beauty Transgene.

Dual reporter Sleeping Beauty transposon system designed for FACS enrichment of K562 cells with DsRed transgene expression. (A) Schematic of the dual fluorescent cis SB-Tn plasmid showing the erythroid specific IHK hybrid promoter consisting of two small enhancer elements, i8 from the human ALAS2 intron 8 and HS40 from human α-globin locus control region, linked to the human ANKYRIN-1 promoter for the red fluorescent protein, DsRed. On the vector backbone external to the SB-Tn’s IR/DRs, the ubiquitous eukaryotic initiation factor 4A1 (eIF) drives a dual expression cassette of SB10 transposase followed by an internal ribosome entry site (IRES) and enhanced green fluorescent protein (GFP) that was utilized in the selection of the transiently transfected cells by fluorescent activated cell sorting (FACS). (B) Fluorescence images two days after transfection with the eIF-SB10-IRES-GFP//pT2-IHK-DsRed showed K562 cells expressing DsRed (left), GFP (right), respectively, and both (middle) with different shades of yellow color when merged. (C) FACS analysis of the transiently transfected cells at two days post-transfection (top panel) using GFP selection. The thirty thousand GFP+ cells selected by FACS were cultured, passaged under normal conditions, and subjected to FACS analysis for tracking DsRed and GFP expression at 1, 2, 4 and 8 weeks post-sorting (lower panels). The culture time in weeks after initial selection for GFP is shown on the right side, and the identity of the cells analyzed is indicated at top of the FACS panels.

Jianhui Zhu, et al. Biochemistry. ;49(7):1507.

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