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Gene Splicing by Overlap Extension or gene SOEing is a PCR-based method of recombining DNA sequences without reliance on restriction sites and of directly generating mutated DNA fragments in vitro. By modifying the sequences incorporated into the 5-ends of the primers, any pair of polymerase chain reaction products can be made to share a common sequence at one end. Under polymerase chain reaction conditions, the common sequence allows strands from two different fragments to hybridize to one another, forming an overlap. Extension of this overlap by DNA polymerase yields a recombinant molecule. This powerful and technically simple approach offers many advantages over conventional approaches for manipulating gene sequences.

SummaryGerminal center (GC) B cells cycle between the dark zone (DZ) and light zone (LZ) during antibody affinity maturation. Whether this movement is necessary for GC function has not been tested. Here we show that CXCR4-deficient GC B cells, which are restricted to the LZ, are gradually outcompeted by WT cells indicating an essential role for DZ access. Remarkably, the transition between DZ centroblast and LZ centrocyte phenotypes occurred independently of positioning. However, CXCR4-deficient cells carried fewer mutations and were overrepresented in the CD73+ memory compartment. These findings are consistent with a model where GC B cells change from DZ to LZ phenotype according to a timed cellular program but suggest that spatial separation of DZ cells facilitates more effective rounds of mutation and selection. Finally, we identify a network of DZ CXCL12-expressing reticular cells that likely support DZ functions.

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The splenic marginal zone (MZ) is a unique microenvironment where resident immune cells are exposed to the open blood circulation1,2. Despite its importance in responses against blood-borne antigens, lymphocyte migration in the MZ has not been intravitally visualized due to challenges associated with achieving adequate imaging depth in this abdominal organ. Here we develop a 2-photon microscopy procedure to study MZ and follicular (FO) B cell movement in the live spleen. We show that MZ B cells are highly motile and exhibit long membrane extensions. MZ B cells shuttle between MZ and follicles with at least one fifth of the cells exchanging between compartments per hour, a behavior that explains their ability to rapidly deliver antigens from the open blood circulation to the secluded follicles. FO B cells also transit from follicles to MZ but unlike MZ B cells, they fail to undergo integrin-mediated adhesion, become caught in fluid flow and are carried into the red pulp. FO B cell egress via the MZ is sphingosine-1-phosphate receptor-1 (S1PR1)-dependent. This study shows that MZ B cells migrate continually between MZ and follicles and establishes the MZ as a site of S1PR1-dependent B cell exit from follicles. The work also shows how adhesive differences of closely related cells critically influences their behavior in the same microenvironment.

Gene Splicing by Overlap Extension (gene SOEing) provides a powerful method of recombining sequences without depending on restriction sites or ligase, and a simple, generally applicable way of using polymerase chain reaction (PCR) to perform site-directed mutagenesis in vitro. This technique allows even those with minimal molecular biology expertise to generate quickly genetic constructs that might otherwise have been impractical using only the older (restriction enzymebased) technology.

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