This was followed by deletion of a neomycin resistance gene (cDNA transfection into targeted ES cells to avoid unpredictable Cre activity23. of the CD19-Cre mice regarding deletion efficiency in IL10-floxed mice. Thus, the CD19-iCre line is a valuable tool for highly efficient gene targeting specific to the B cell compartment. sites (termed as floxed). These Cre driver lines (CD19-Cre and Mb1-iCre) have been well characterized and used to successfully assess the function of genes through their deletion or genetic manipulation in a B cell-specific manner10,11. However, Cre-mediated deletion in the CD19-Cre and Mb1-iCre lines exhibits flaws with regard to its efficiency and specificity, respectively. When B cell-specific Cre mice were crossed with mice harboring the Rosa26-enhanced yellow fluorescent protein (EYFP) reporter (R26EYFP+/fl; hereafter referred to as mice)12 which express EYFP directly from the ubiquitous promoter following Cre-dependent deletion of a (deletion in bone marrow B cells, including in pro-B cells, when crossed with mice11,13. Thus, researchers often use the Mb1-iCre line for the deletion or manipulation of a gene to analyze early B cell development in the bone marrow. With respect to peripheral tissues such as the spleen and lymph nodes, both and Mb1-iCre/Rosa26-EYFP (reporter mice, there can be a small fraction of EYFP+ T cells detected in the Mb1-iCre line, suggesting minor aberrant knock-in line (CD19-iCre), in which the sequence is inserted in-frame to the 3 end of the coding sequence (immediately upstream of the stop codon). By crossing with the line, we show that the CD19-iCre driver line enables highly efficient and specific ablation of floxed genes in the B cell lineage from the pro-B cell stage without infidelity of Cre-mediated recombination in T cells. When crossed with IL10-floxed mice, the CD19-iCre line was found to be superior to CD19-Cre mice with regard to the disruption of IL-10 production in B cells. Thus, the CD19-iCre line provides a new option for generating B cell-specific deficient mice with high specificity and efficiency, which further facilitates investigating the functional roles of a gene throughout B lineage cells. Results Generation of the CD19-iCre knock-in mice To generate a mouse line that expresses the iCre recombinase20 in CD19-expressing B lineage cells without disturbing its endogenous expression, we GSK2200150A generated a CD19-T2A-iCre knock-in line, in which the T2A-iCre sequence was inserted in-frame to the 3-end of the coding sequence (immediately upstream of the stop codon) (Fig.?1a). Following the induction of double-strand DNA breaks by the Cas9 enzyme mediated by a guide RNA (gRNA), the homology arms GSK2200150A guided the T2A-iCre to be inserted in-frame with the open reading frame by homologous directed repair. This targeting strategy using self-cleaving 2A sequences21 was designed to mediate bicistronic translation, which is potentially more efficient than the well-described internal ribosome entry site sequence. Upon translation of the chimeric mRNA, the sequence led to ribosome skipping22, resulting in the co-expression of CD19 and iCre as discrete proteins in CD19-expressing cells. Consequently, iCre expression was regulated by the promoter in tandem with the endogenous CD19 expression. In the present study, the targeting construct was correctly GSK2200150A introduced in embryonic stem (ES) cells, which was confirmed by polymerase chain reaction (PCR) analysis. This was followed by deletion of a neomycin resistance gene (cDNA transfection into targeted ES cells to avoid unpredictable Cre activity23. After confirming the Neo cassette deletion by PCR, the targeted ES cells were transferred into blastocysts and the mouse germline (Fig.?1b). Heterozygous and homozygous CD19-iCre mice (referred to as locus. Neomycin resistance gene cassette (Neo) is flanked by sites and inserted downstream of the untranslated region. The KI allele is obtained after Flippase (Flp) site-directed recombination of the selection marker. (b) PCR for detection of transgene KI allele with genomic DNA from wild-type (WT), heterozygous and homozygous CD19-iCre mice. Amplicons of 484 and 247?bp are identified by primer pairs (p1Cp2 and p1Cp3) specific for Rabbit Polyclonal to CCDC102A the WT and iCre alleles, respectively. (c) Flow cytometry of the surface expression of CD19 on B220hi B cells of peripheral blood from WT, homozygous and heterozygous Compact disc19-Cre and Compact disc19-iCre mice. Best, mean fluorescence strength (MFI) from the staining of Compact disc19. (d) Quantitative RT-PCR of mRNA encoding Compact disc19 in WT and Compact disc19B cells, normalized towards the appearance of -actin. (e) Traditional western blotting evaluation of whole-cell lysates of splenic B cells from WT, Compact disc19mglaciers with antibodies particular for Compact disc19, Cre, and -actin. The arrow and arrowheads indicate fusion proteins.