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. 2011 Jun 14;286(31):27123–27131. doi: 10.1074/jbc.M111.230995

FIGURE 1.

FIGURE 1.

Bacterial artificial chromosome construction and transgenesis. A, diagram of the IgHΔ3alx3b BAC before and after hs3b deletion. Upper map, CHC BAC, original BAC containing ∼230 kb derived from the Igh locus of the 129 mouse strain. DH, DHQ52 gene segment proximal to the JH region (JH1–4). Gray boxes denote the heavy chain constant region (CH) coding segments (μ, γ3, etc.); open ovals denote regulatory regions Eμ and 3′ regulatory region (3′RR). 3′RR consists of seven DNase I hypersensitivity regions (3a, 1,2, etc.) Maps not drawn to scale. Center map, IgHΔ3alx3b (JH gene segments replaced with B1–8 VH gene; hs3a deleted; black triangles denote loxP sites surrounding hs3b; 397-bp DNA tag (TAG)). Lower map, IgHΔ3aΔ3b (loxP-mediated deletion of hs3b). B, restriction enzyme maps and Southern blots of CHC and IgHΔ3alx3b BACs in the region of VH insertion. Maps are above, and Southern blot is below. J1-J4, JH gene region; open oval, Eμ regulatory region; VDJ, inserted VH region; IgH BAC, IgHΔ3alx3b. E = EcoRI, B = BamHI, H = HindIII. Black rectangle under each map and downstream of JH region denotes DNA probe used for Southern blot below. Southern blot is of CHC and IgHΔ3alx3b (IgH) BAC DNAs. Restriction enzymes are indicated below each pair of lanes. Molecular weight markers (in kb) are indicated to the left. Expected fragment sizes are: BamHI (6.6-kb CHC, 8.5-kb IgH), EcoRI (6.2-kb CHC, 7.0-kb IgH), HindIII (2.3-kb CHC, 6.0-kb IgH). C, BAC structure (hs3b region) in transgenic mice. Southern blot is above, and maps are below. Liver DNAs were digested with HindIII (H) and hybridized to the 3bD probe (black box below maps). C57BL/6J DNA was included as a control for genetic background (endogenous Igh loci of transgenic mice). 69, 60, 79, and 83 denote four transgenic lines prior to hs3b deletion; Δ69, etc. denote matched lines after hs3b deletion. Expected HindIII fragment sizes are: 8.1 kb (C57-derived hs3b fragment); 3.8 kb (from BAC, before hs3b deletion); 4.2 kb (from BAC, after hs3b deletion). In the maps, H = HindIII. Gray box denotes tag DNA; black arrowheads denote loxP sequences.