calponin-3 in the S2 Schneider cell system. S2 cells were transfected with expression constructs encoding calponin-3 and Syk, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19713189 Lyn or Btk, respectively. Cellular lysates were subjected to SDS-PAGE and western blotting. E. Confocal image of pre-B cells expressing GFP or a calponin-3-GFP fusion protein, respectively. F. Western blot for analysis of calponin 2 and 3 expression in bone marrow cells cultured with IL-7 for 5d, in sorted CD19- and CD19+ splenic B cells, in total thymocytes and in the brain. Western blotting against actin was used as a loading control. doi:10.1371/journal.pone.0128385.g001 6 / 16 Calponin-3 in B Lymphocyte Development Fig 2. Targeting of ES cells to generate a floxed calponin-3-GFP knock-in. A. Schematic illustration of the Cnn3 locus, the targeting construct and the Cnn3 locus after targeting. The targeting strategy aimed at replacing exons 2 to 5 
with a floxed mini gene corresponding to exons 2 to 7 fused to a GFPcDNA. Exons and the mini gene are represented by grey boxes, the neomycin-resistance gene PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19709857 is illustrated by a white box. LoxP-sites are purchase UNC0642 depicted as black triangles, FRT-sites as white triangles. Restriction sites for the enzymes used for southern blot analysis are indicated. Please note that the illustration is not in scale. B. Southern blot analysis of the targeted clone used for blastocyst injection. Genomic DNA was digested by NcoI, HindIII, BamHI or KpnI, respectively, separated by agarose gel electrophoresis and blotted. The blot was hybridized with the internal 5′- and the external 3′-probe as indicated in A. Arrows indicate the positions of fragments corresponding to the non-targeted as well as the targeted allele. doi:10.1371/journal.pone.0128385.g002 was constructed where Cnn3 exons 2 to 5 were replaced by a floxed mini gene comprising Cnn3 exons 2 to 7, deleted for the stop codon and fused to a GFP cDNA. Following splicing from the endogenous exon 1 to the mini gene, this was expected to result in expression of a floxed full-length calponin-3-GFP fusion protein under control of the endogenous promoter and its regulatory elements. This strategy allowed for a dual application: First, conditional Cre-mediated deletion of the mini gene generates a null allele, enabling the tissue-specific analysis of cellular function in the absence of calponin-3. Second, targeted mice serve as a fluorescent reporter to track cells and tissues for calponin-3 expression in vivo. Of note, a corresponding calponin-3-GFP fusion protein was tested beforehand in pre-B cells and displayed the same pervanadate-induced phosphorylation as the HA-tagged calponin-3, suggesting that the C-terminal GFP tag does not compromise protein function. A 129S6/SVEvTac ES cell line was transfected with the targeting vector, and from about 580 clones growing under G418 selection, 6 clones were positive for the correct integration by Southern blot analysis with a 3′ external probe. However, detailed analysis by additional Southern blot hybridizations, PCR and sequencing revealed that only one of these clones was 7 / 16 Calponin-3 in B Lymphocyte Development correctly targeted at the 5′ end and contained the 5′ loxP site. When injected into C57BL/6 blastocysts, this clone produced chimeric mice that were crossed with FLPe-transgenic mice to remove the gene encoding neomycin resistance. Germ line transmission of the knock-in and deletion of the neo gene were confirmed by PCR. Heterozygous intercrosses of calponin-3-GFP mice generated the e