1. Volume 2, Issue 4, Pages 447-455 (October 1998)
The β-Globin LCR Is Not Necessary for an Open Chromatin Structure or Developmentally Regulated Transcription of the Native Mouse β-Globin Locus 
Elliot Epner, Andreas Reik, Daniel Cimbora, Agnes Telling, M.A Bender, Steve Fiering, Tariq Enver, David I.K Martin, Marion Kennedy, Gordon Keller, Mark Groudine 
Molecular Cell 
Volume 2, Issue 4, Pages (October 1998)
DOI: /S (00)

2. Figure 1
Creation of the ΔLCR Chromosomes Using Homologous and Site-Specific Recombination
(A) Strategy for the creation of the ΔLCR mouse chromosomes using gene targeting, step-up, and site-specific recombinases as described in the text and Experimental Procedures. ΔLCR clones were confirmed by Southern blotting as shown in (B). The LCR deletion was made in either a nonerythroid (ES) or an erythroid (K562 cells) environment by expressing Cre recombinase in either cell type. Hatched boxes represent loxP sites. HpaI sites in the LCR are indicated by H, and the probe used for the Southern analysis in (B) is shown.
(B) Southern analysis of ES cells carrying mutations in the β-globin LCR. DNA from wild-type, LCR deleted, and various intermediates was cut with HpaI, fractionated by agarose gel electrophoresis, blotted, and probed with a fragment (Hpa-Kpn) located 5′ to mouse 5′ HS6. Using this probe/enzyme combination, novel and diagnostic fragments are observed in the Δ1,Δ5–6neo and Δ1–6 samples, as shown.
Molecular Cell 1998 2, DOI: ( /S (00) )

3. Figure 2
General DNaseI Sensitivity of the Mouse β-Globin Locus after Deletion of the LCR in K562 Cells
ES cells with the Δ1,Δ5–6neo genotype were transferred into K562 cells by microcell hybrid-mediated transfer, and Cre recombinase was transiently expressed to delete the entire LCR and the selectable marker gene. Nuclei were digested with increasing concentrations of DNaseI. DNA was purified, digested with EcoRI, fractionated by agarose gel electrophoresis, and blotted. Each lane contains 15 μg of DNA. Filters were hybridized with various mouse (m) and human (h) probes, washed, and autoradiographed. Mouse restriction fragments: mβmaj is a 7 kb EcoRI fragment containing the mouse βmajor gene; m5′Ey is a 2 kb EcoRI fragment 3′ to 5′HS1. Human restriction fragments: hψβ is a 7 kb fragment containing the human ψβ gene; hcoll corresponds to 4 and 2.8 kb EcoRI fragments containing the human α2 collagen gene; h3′β is a 3.6 kb EcoRI fragment containing the third exon and sequences 3′ to the human β-globin gene. (See Experimental Procedures for probe details.)
Molecular Cell 1998 2, DOI: ( /S (00) )

4. Figure 3
General DNaseI Sensitivity of the Mouse β-Globin Locus after Deletion of the LCR in ES Cells and Transfer of the ΔLCR Chromosome from ES to K562 Cells
General DNaseI sensitivity of (A) wild-type and ΔLCR mouse β-globin loci in ES cells and (B) the mouse β-globin locus after deletion of the LCR in ES cells and transfer into K562 cells. DNA was purified from DNaseI-digested ES and K562 nuclei, digested with BamH1, fractionated by agarose gel electrophoresis, and blotted. Each lane contains 15 μg of DNA. (A) Filters were hybridized with probes from the mouse β-globin and cyclin D1 (cycΔ1) loci and autoradiographed. Mouse β-globin restriction fragments: mβ 3.5 kb is a fragment containing sequences 3′ to the βh1 gene; mβ 3.2 kb is a fragment containing the Ey gene (see Experimental Procedures for probes). (B) The chromosome containing the ΔLCR mouse β-globin locus was marked at the cyclin D1 locus by gene targeting in ES cells and then transferred into K562 cells by microcell-mediated transfer. Filters were hybridized with mouse (m) and human (h) probes, washed, and autoradiographed. Mouse restriction fragments: mmyoD is a 10.5 kb BamH1 fragment containing the mouse myoD1 gene; m3′βmaj is a 7.4 kb BamH1 fragment containing the second intron and third exon of the mouse βmajor gene and sequences 3′ to it. h3′β is a 10 kb BamH1 fragment containing the second intron and third exon of, as well as sequences 3′ to, the human β-globin gene (see Forrester et al. 1986, Forrester et al. 1990, Forrester et al. 1994; Epner et al for probes).
Molecular Cell 1998 2, DOI: ( /S (00) )

5. Figure 4 Analysis of β-Globin Transcription in K562 Cells
A K562 cell line containing a wild-type (wt) mouse chromosome 7, two lines into which the ΔLCR mouse chromosome was introduced from ES cells, and a K562 cell that contains no mouse chromosomes (−) were induced with hemin. RNA was prepared, and RT-PCR analysis performed with a primer pair that coamplifies human γ and mouse βh1 RNA or a primer pair that coamplifies human ε and mouse Ey RNA (see Experimental Procedures); the coamplified products are distinguished by mouse-specific restriction enzyme sites. The human signals were used as internal controls for RNA input and RT-PCR efficiency. RNA from in vitro differentiated ES cells was analyzed to provide a control for mouse β-globin signals; these cells express both embryonic and adult globin. Bands were quantitated by phosphorimager analysis, and the murine-to-human signal ratio was calculated and standardized in relation to the wild-type ratio that was set to 100. The obtained values are shown below the autoradiographs.
Molecular Cell 1998 2, DOI: ( /S (00) )

6. Figure 5
Single-Cell Analysis of Mouse and Human β-Globin Expression in Erythroid Hybrids
(A) Autoradiogram of RT-PCR products from single K562 hybrid cells carrying the wild-type (wt) mouse chromosome. Human γ and mouse βh1 products, distinguished by restriction digest and gel electrophoresis, are indicated. Results from ten representative cells are presented. The βh1/γ ratio, calculated after quantitation by a phosphorimager, is indicated for each cell.
(B) Results from ten representative K562 hybrid cells (clone #1) carrying the mouse chromosome 7 from which HS1–6 were deleted prior to transfer from ES to K562 cells.
(C) Summary of results from all single cells analyzed. Ratios of βh1 to γ expression in single cells carrying the wt mouse chromosome and the ΔLCR chromosome are represented by bars. Asterisk denotes those cells in which no detectable βh1 signal was obtained.
Molecular Cell 1998 2, DOI: ( /S (00) )

7. Figure 6 Analysis of β-Globin Transcription in ES Cells
(A) Wild-type and ΔLCR ES cells were removed from LIF and differentiated in vitro (see Experimental Procedures). At days 7 and 10 after removal from LIF, RNA was prepared from the differentiated cells, and RT-PCR analysis was carried out with the indicated combinations of primers from the murine β-globin locus and an α-globin-specific primer pair.
(B) Primitive and definitive erythroid colonies from homozygous wtLCR and homozygous ΔLCR EBs. Primitive colonies (EryP) were cultured for 4 days and definitive (EryD) colonies for 8 days. Original magnification for primitive colonies was 320× and for definitive colonies was 200×.
(C) Globin RNA analysis of erythroid and macrophage colonies. Primitive erythroid colonies were harvested at day 4 of culture, definitive erythroid colonies at day 8 of culture, and macrophage colonies at day 7 of culture. Lysates of individual colonies were subjected to reverse transcription, tailing, and PCR amplification (see Experimental Procedures for details), and amplified products were Southern blotted and hybridized with the indicated probes. The “controls” panel includes hybridization of indicated probes to amplified products from primitive erythroid (Ep) and macrophage (MAC) colonies grown from day 6 EB precursors, and from definitive erythroid (Ed) colonies derived from fetal liver, as well as from “mock” PCR reactions performed in the absence of any colonies. Expression of the ribosomal L32 gene was used as an indication of the amount of material in each lane.
Molecular Cell 1998 2, DOI: ( /S (00) )