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Results: 7

1.
Figure 6

Figure 6. From: Reproducible doxycycline-inducible transgene expression at specific loci generated by Cre-recombinase mediated cassette exchange.

Stability of gene induction at the LoxP loci. Histogram plots showing GFP induction in M2K #3 and M2PK #1 before and after 1 month of continuous passage. Experiments were performed twice and data shown are from one experiment.

Ee Tsin Wong, et al. Nucleic Acids Res. 2005;33(17):e147-e147.
2.
Figure 7

Figure 7. From: Reproducible doxycycline-inducible transgene expression at specific loci generated by Cre-recombinase mediated cassette exchange.

Monitoring cell doubling using inducible H2BGFP. HeLa cells stably expressing TRE-H2BGFP were generated by L3-2L mediated RMCE. Cells were either not induced (− dox) or induced with 2 µg/ml dox (+ dox) for 3 days to achieve maximum expression before washing out the dox in the presence or absence of 2 mM thymidine. Cells were harvested each day after the removal of dox and their fluorescence monitored by flow cytometry (Actual fluorescence). The expected fluorescence is the theoretical fluorescence value assuming 50% decay after each population doubling. Experiments were performed twice and data from one experiment are shown as a histogram in (a) and as a graph in (b).

Ee Tsin Wong, et al. Nucleic Acids Res. 2005;33(17):e147-e147.
3.
Figure 1

Figure 1. From: Reproducible doxycycline-inducible transgene expression at specific loci generated by Cre-recombinase mediated cassette exchange.

Principle of RMCE and screening strategy for recombinants. The positive-negative selection marker HyTK that confers resistance to hygromycin (HygR) and sensitivity to ganciclovir (GanS) is flanked by a pair of heterospecific LoxP sites placed in inverted orientation (inverted triangles) and integrated as a single copy into a genomic site. In the presence of Cre-recombinase and incoming DNA harboring the same pair of LoxP sites, exchange will replace the existing HyTK with DNA on the exchange plasmid. Cells that have successfully undergone RMCE can be selected based on resistance to ganciclovir. The tetracycline inducible LucGFP (TRE-LucGFP) is used as the reporter to characterize uniformity of gene expression between clones after integration into the genome. Also shown are the positions of BamHI site and Luc probe used for Southern blot and primer pairs (P1–P6) used for screening of recombinants.

Ee Tsin Wong, et al. Nucleic Acids Res. 2005;33(17):e147-e147.
4.
Figure 5

Figure 5. From: Reproducible doxycycline-inducible transgene expression at specific loci generated by Cre-recombinase mediated cassette exchange.

Reporter gene induction by two plasmids Tet-On stable HeLa M2K RMCE clones. (a) Schematic diagram showing the various genes that were incorporated to make up M2K RMCE clones. Two independent promoters are used to drive the rtTA and tTR genes. The reporter gene LucGFP is inserted into the genome by RMCE. (b) Normalized luciferase activity to show maximum gene induction in cells before and after incubation with 2 µg/ml dox for 72 h. Data shown are mean with standard error obtained from three independent experiments. Parental line (P) do not express the luciferase gene. (c) Histogram plots of GFP expression by M2K RMCE derivatives before (−) and after (+) dox induction at 0.1 µg/ml for 72 h. Cells were harvested for flow cytometry and each population was gated (M1 and M2) with the corresponding percentage of gated cells and mean GFP fluorescence shown in each plot. (d) Graph shows mean GFP fluorescence of RMCE clones obtained from two independent RMCE experiments. Cells were either un-induced (−) or treated with 0.1 µg/ml dox for 72 h.

Ee Tsin Wong, et al. Nucleic Acids Res. 2005;33(17):e147-e147.
5.
Figure 2

Figure 2. From: Reproducible doxycycline-inducible transgene expression at specific loci generated by Cre-recombinase mediated cassette exchange.

Construction and characterization of new LoxP site for RMCE. (a) Sequence of the 8 bp spacer found within the LoxP site. LoxP and LoxP511 are named according to previously published (12). Recombination occurs efficiently between LoxP sites with identical spacer sequence. Changes to the sequence within the 8 bp spacer generates LoxP sites that recombine with themselves but not with other LoxP sites. Underlined base(s) represents the base change that differs from the original L2 sequence. (b) A schematic of the three test plasmids (or I = input DNA), pL1L2, pL3L3 and pL32L and the possible products of RMCE (R = recombinant). Shown are the positions of the LoxP sites, the restriction sites used for the Southern blot analysis (S = Ssp I, N = Nde I), and the target sequence for the probe (black box). The sizes of the predicted restriction fragments are listed below. The boxed fragment corresponds to that detected by the probe. Note that only the L3L3 intramolecular recombination would be predicted to occur if L1, L2 and L3 do not recombine with each other. (c) Southern blot analysis of test plasmids transfected into 293 cells in the absence or presence of a Cre-expressing plasmid [pOG231, (14)]. Bands were detected using a probe to the hygromycin sequence common to the test plasmids. Unrecombined, input plasmids are marked with ‘I’; products of Cre recombination are marked with ‘R’. Note the presence of a non-specific band in lanes 3 and 5 marked with an asterisk.

Ee Tsin Wong, et al. Nucleic Acids Res. 2005;33(17):e147-e147.
6.

Figure 4. From: Reproducible doxycycline-inducible transgene expression at specific loci generated by Cre-recombinase mediated cassette exchange.

GFP induction by single plasmid Tet-On CHO 111-134 and HeLa M2PK RMCE clones. The RMCE clones were named after the L3-2L parent that they were derived from. (a) Schematic diagram showing the various genes that were incorporated to make up the RMCE clones. In CHO 111-134 and HeLa M2PK, rtTA and tTR genes are linked by IRES and transcriptionally driven by one promoter. NeoR codes for neomycin/G418 resistance gene. The L3-TRE-LucGFP-2L cassette is inserted as single copy into the genome by RMCE as described in Figure 1. (b) Histogram plot showing green fluorescence exhibited by CHO 111-134 after induction with increasing concentration of dox for 72 h. (c) Graph shows the mean fluorescence units exhibited by CHO 111-134 after induction with increasing dox concentrations for 72 h. Data are obtained from two independent experiments. (d) Normalized luciferase activity to show maximum gene induction in cells before and after incubation with 2 µg/ml dox for 72 h. Data represents mean and standard error of three independent experiments. Parental lines (P) do not have luciferase gene inserted into their genomes. (e) Histogram plots of GFP expression by RMCE derivatives before (−) and after (+) dox induction in one representative experiment. HeLa and CHO cells were induced with 0.1 and 0.05 µg/ml dox, respectively for 72 h before harvesting for flow cytometry. Each population was gated (M1 and M2) and the percentage of the gated cells and their corresponding mean GFP fluorescence are indicated in each plot.

Ee Tsin Wong, et al. Nucleic Acids Res. 2005;33(17):e147-e147.
7.
Figure 3

Figure 3. From: Reproducible doxycycline-inducible transgene expression at specific loci generated by Cre-recombinase mediated cassette exchange.

Integration and analysis of L3-2L introduced into the genomes of CHO and HeLa cells. (a) Engineering L3-2L into DR-8 cell line by homologous recombination using the strategy described by Kalejta et al., (17). The diagram shows the genomic structure at the DHFR locus (wild-type), sizes of the fragments generated by EcoRI digestion and the positions of the probes used for Southern blot. Targeting cosmid DNA containing the 3′ half of the DHFR gene is used to reconstruct the entire gene in DR-8 cell line that contains truncation at the 3′ end of the DHFR gene. The L3 and L2 sites are placed in inverted orientation and flanking the HyTK positive-negative selectable marker gene. The entire cassette was inserted downstream of DHFR gene after homologous recombination. (b) Southern blot of genomic DNA derived from CHO cells hemizygous for wild-type DHFR [WT/- or UA21 (17)], 3′ truncated DHFR [delta/- or DR-8 (17)] and HyTK reconstituted cells (HyTK/- or 146-111). DNA was restricted with EcoRI before blotting and the positions of the bands generated by each probe are indicated with an arrow. Probe 121 marks the 5′ flank of the cosmid DNA, probe 100 tests for reconstruction at the 3′ end of the DHFR gene, probe 12/38 indicates the presence of ori-beta (β), probe eight marks the 3′ flank of the cosmid DNA, and probe HyTK indicates the insertion of HyTK into the genome. M, molecular weight marker. (c) Schematic diagram of retrovirus RV-L3HyTK2L. L3 and L2 are placed in inverted orientation flanking HyTK gene. Also shown is the position of the BamHI site and the probe used for Southern blot. Upon integration into the genome, the expected size of the fragment generated by BamHI is 4 + x kb. (d) Southern analysis to determine the copy number of integrated HyTK gene in the HeLa cell clones (1 to 10) derived after infection with RV-L3HyTK2L. Genomic DNA was restricted with BamHI and probed against HyTK gene. Positive control (+) is original retrovirus DNA used to generate the stable line. P, parental HeLa.

Ee Tsin Wong, et al. Nucleic Acids Res. 2005;33(17):e147-e147.

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