1. β-globin Gene Switching and DNase I Sensitivity of the Endogenous β-globin Locus in Mice Do Not Require the Locus Control Region 
M.A Bender, Michael Bulger, Jennie Close, Mark Groudine 
Molecular Cell 
Volume 5, Issue 2, Pages (February 2000)
DOI: /S (00)

2. Figure 1 Generation of Mice Lacking the LCR
(A) Strategy for deleting the LCR. Multiple targeted deletions were performed in ES cells, resulting in deletion of 5′HS 1, 4, and 5–6 and flanking the LCR with loxP sites. In addition, a 3′ segment of neo (EO) was added to facilitate adding sequences back to the locus. Mice with the resultant Δ1Neo, Δ4ΔNeo, Δ5–6 Hyg EO genotype (Δ145) were bred with Cre-expressing mice to generate ΔLCR mice lacking 5′HS 1–6. Numbers 1–6 designate 5′HS 1–6, respectively. H, HpaI sites used for mapping. Shaded boxes with an overlying L indicate loxP sites. Hyg and Neo represent PGK-hygromycin and PGK-neomycin phosphotransferase selectable markers, respectively.
(B) Southern blot analysis of mice carrying deletions of the β-globin LCR. DNA from mice with the designated genotypes was digested with HpaI and hybridized with a probe spanning −33,787 to −33,151 relative to the Ey cap (see A). D, wild-type HbbD allele. Other genotypes as above.
Molecular Cell 2000 5, DOI: ( /S (00) )

3. Figure 2 Phenotype of ΔLCR Heterozygotes
Wildtype D/S and ΔLCR-D/S embryos at dpc 15.5.
Molecular Cell 2000 5, DOI: ( /S (00) )

4. Figure 3
Developmental Expression of β-like globin Genes in ΔLCR Heterozygous Mice
The HbbD allele (D) is either wild type or carries the targeted mutation, while the HbbS allele (S) is wild type. RNA was analyzed from dpc 10.5 and 12.5 yolk sac, dpc 12.5 and 15.5 fetal liver, and adult peripheral blood using RT-PCR assays for Ey, βh1, and adult primers (top, middle, and bottom panels). WT, wild-type D/S animals. ΔLCR, mutant ΔLCR-D/S heterozygous mice. S and D mark the RT-PCR products from the S and D alleles, respectively.
Molecular Cell 2000 5, DOI: ( /S (00) )

5. Figure 4
General DNase I Sensitivity and Hypersensitive Site Formation after Deletion of the LCR
Nuclei from splenic erythroid cells from ΔLCR homozygous mice rescued with a human β-globin-containing YAC or from wild-type mice were isolated and digested with varying amounts of DNase I. The resultant DNAs were digested with BamHI to completion (EcoRV for 3′HS 1), Southern blotted, and hybridized with the probes indicated. The first lane demonstrates the intact parent band followed by samples having undergone increasing DNase I digestion.
(A) General DNase I sensitivity of the embryonic globin region in ΔLCR homozygous mice. α1 AT, a liver-specific DNase I–resistant control compared to a similar sized fragment from the 3′ end of the endogenous mouse βh1-globin gene.
(B) General DNase I sensitivity of the embryonic globin region in wild-type mice. Probes and sizes as in (A).
(C) General DNase I sensitivity of the adult region in ΔLCR homozygous mice. α1 AT, a liver-specific DNase I–resistant control compared to a smaller sized fragment from between the mouse βmaj and βmin genes.
(D) Mapping of mouse β-globin HSs in the absence of 5′HS 1–6. Top panel, the mouse βmaj promoter. The 5′ m βmaj probe hybridizes upstream of the promoter, yielding a 4.7 kb parent band and a degradation band that maps to the promoter region. Bottom panel, 3′HS 1. The 3′HS 1 probe yields a 6.1 kb parent band and a doublet of degradation bands characteristic of 3′HS 1.
Molecular Cell 2000 5, DOI: ( /S (00) )