As shown inFig. hs1.2 and hs4, although individually dispensable for CSR, are, together, sufficient to support CSR. Keywords:Antibodies, DNA Recombination, Gene Expression, Gene Regulation, Immunology, Lymphocyte, Transcription Enhancers, Class-switch Recombination, Immunoglobulin Genes, Transgenic Mice == Introduction == The immunoglobulin heavy and light chain (IgH and IgL) loci undergo multiple rounds of DNA recombination and somatic hypermutation as B lymphocytes form and then perfect their receptors for antigen. The process of heavy chain class-switch recombination (CSR)3takes place upon antigen activation of the B cell and allows for the development of child clones that can Ibuprofen (Advil) produce antigen-reactive antibody with the appropriate properties to combat the pathogen in question (e.g.ability to traffic to appropriate sites in the body; ability to directly eliminate the organism through complement-mediated lysis; etc.). Although activation-induced cytidine deaminase has been identified as an essential protein, central to both CSR and the somatic hypermutation of variable-region encoding genes (1), the means by which activation-induced cytidine deaminase activity is usually recruited to the appropriate DNA targets within the Ig loci remains largely unknown. Several studies have shown that this 3 regulatory region (3RR) plays an important role in class-switch Ibuprofen (Advil) recombination, implicating it in this recruitment process (25). The 3RR is usually a series of DNase hypersensitivity sites, in the beginning identified as enhancer-like elements, that map downstream of the Ig heavy chain constant region genes (611) (seeFig. 1A). Analogous elements have been explained downstream of each of the two C genes in the humanIghlocus (12,13), arguing that their function is usually evolutionarily conserved. == FIGURE 1. == Bacterial artificial chromosome construction and Ibuprofen (Advil) transgenesis.A, diagram of the IgH3alx3b BAC before and after hs3b deletion.Upper map,CHC BAC, initial BAC containing 230 kb derived from theIghlocus of the 129 mouse strain.DH, DHQ52 gene segment proximal to the JHregion (JH14).Gray boxesdenote the heavy chain constant region (CH) coding segments (, 3, etc.);open ovalsdenote regulatory regions E and 3 regulatory region (3RR). 3RR consists of Ibuprofen (Advil) seven DNase I hypersensitivity regions (3a, 1,2, etc.) Maps not drawn to level.Center map, IgH3alx3b (JHgene segments replaced with B18 Ibuprofen (Advil) VHgene; hs3a deleted;black trianglesdenote loxP sites surrounding hs3b; 397-bp DNA tag (TAG)).Lower map, IgH3a3b (loxP-mediated deletion of hs3b).B, restriction enzyme maps and Southern blots of CHC and IgH3alx3b BACs in the region of VHinsertion. Maps are above, and Southern blot is usually below.J1-J4, JHgene region;open oval, E regulatory region;VDJ, inserted VHregion;IgH BAC, IgH3alx3b.E= EcoRI,B= BamHI,H= HindIII.Black rectangleunder each map and SPN downstream of JHregion denotes DNA probe utilized for Southern blot below. Southern blot is usually of CHC and IgH3alx3b (IgH) BAC DNAs. Restriction enzymes are indicated below each pair of lanes. Molecular excess weight markers (in kb) are indicated to theleft. 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 boxbelow maps). C57BL/6J DNA was included as a control for genetic background (endogenousIghloci of transgenic mice).69,60,79, and83denote four transgenic lines prior to hs3b deletion; 69, 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 boxdenotes tag DNA;black arrowheadsdenote loxP sequences. Class-switch recombination is usually preceded by transcription from intronic promoters that lie upstream of the constant region genes. These transcripts are termed germline (GLT) as they are generated.