Figure 1.
Schematic presentation of the human β-globin locus in PAC transgenic constructs.
A) Arrows on top of the locus depict the individual hypersensitive sites within the LCR. β-like globin genes are indicated by large triangles with different colors. The β-mark globin gene was indicated by light blue rectangle with βm labeled on top of it. LTR located at the 5′-upstream of the βm gene was indicated by the small rectangle with black in color. The HS5 is flanked by loxP sites indicated by small triangle on the locus. PACΔ constructs were generated by homologous recombination according to Iman et al., 2000. The white boxes indicate the olfactory receptor (OR) genes which flank the β-globin locus. HindIII restriction sites (small arrows) and the DNA fragments used in the 3C analysis (solid rectangles) are shown below the PAC1B locus. Distances are in kb counting from the transcription initiation site of the ε-globin gene. B) Structure of the marked β-globin gene (βm) and the ChIP-βm primers location.
Figure 2.
3C analysis of the PAC1B transgene using a primer from the human β-globin gene in combination with primers from other parts of the locus.
Fetal livers were collected from E14.5 embryos for this set of 3C experiments. (A) Representative examples of the PCR fragments resulting from the 3C experiments. WT: PAC8.1wt; RT: Random template control. (B) Histogram of the relative crosslinking efficiencies after quantitation and normalization. The histograms are the average of at least three separate experiments, with each PCR performed in triplicate.
Figure 3.
3C analysis of the PAC1B and PACΔ1B transgenic lines using a primer from the human β-globin gene in combination with primers from other parts of the locus.
Fetal livers were collected from E14.5 embryos for this set of 3C experiments. (A) Representative examples of the PCR fragments resulting from the 3C experiments. (B) Histogram of the relative crosslinking efficiencies after quantitation and normalization. The histograms are the average of at least three separate experiments, with each PCR performed in triplicate.
Figure 4.
3C analysis of the PAC3K and PACΔ3K transgenes using a primer from 5′HS2 as the fixed primer.
Embryonic blood was collected from E10.5 embryos for this set of 3C experiments. (A) Representative examples of the PCR fragments resulting from the 3C experiments. RT: Random template control. (B) Histogram of the relative crosslinking efficiencies after quantitation and normalization.
Figure 5.
3C analysis of the PAC3K and PACΔ3K transgenes using a primer from the human β-globin gene as the fixed primer.
Embryonic blood was collected from E10.5 embryo for this set of 3C experiment. (A) Representative expamples of the PCR fragments resulting from the 3C experiments. RT: Random template control. (B) Histograms of the relative crosslinking efficiencies after quantitation and normalization.
Figure 6.
CTCF-ChIP assay analysis of the binding of CTCF protein to the mouse HS62 (mHS62), βm and human 5′HS5 in the primitive (A) and definitive (B) stage.
Fold enrichment of the test sequence in bound with CTCF versus input/starting material is shown on the y axis.
Table 1.
Sequence of 3C-PCR primers.
Table 2.
PCR primers used in the Anti-CTCF ChIP Real-time PCR experiments.