c-Kit-Mediated Functional Positioning of Stem Cells to Their Niches Is Essential for Maintenance and Regeneration of Adult Hematopoiesis
Figure 1
Generation of c-Kit conditional KO GFP-reporter mice by employing lox66–71 recombination, enabling tracking of c-Kit-deficient cells.
(A) A targeting vector was designed for the insertion of a complex cassette into the 5′-region of the c-Kit gene. Lox66 and lox71 sites in a head-to-head orientation flank splice acceptor (SA): an inserted internal ribosomal entry site (IRES):eGfp as well as two polyadenylation/stop signals. Prior to Cre recombination, the c-Kit lox66–71 allele will transcribe a full-length c-Kit transcript. Upon activation of Cre, the inserted genes are inverted by the flanking lox66 and lox71. The IRES downstream from the c-Kit exon 8 allows for a bicistronic expression of eGfp and for an activation of stop signals from two polyadenylations, thereby creating a transcription stop of the downstream c-Kit gene. B, BamHI; H, HindIII; K, KpnI; RV, EcoRV; S, SalI; Sb, SbfI; TK, thymidine kinase; X, XbaI. (B) Representative Southern blot screen for ES cells and neo-excised ES clones. XbaI digestion and 3′-flanking probe were used to identify targeted ES cells. The genomic DNAs from the ES clones were digested with BglII, and the blot was probed with the 3′-flanking probe. Expected size of fragments is indicated to the left. Genotypes are indicated above each lane. (C) Schematic strategy to generate c-Kit conditional KO GFP-reporter mice. (D) Representative FACS dot plot showing the lack of c-Kit expression on Lin−cells from BM cells of c-KitW/ΔGFP mice was confirmed by flow cytometric analysis. Left panels show the absence of c-Kit expression on Lin− cells isolated from BMs of c-KitW/ΔGFP mice. Green dots in the Sca-1 and c-Kit profiles gated on Lin− cells represent Lin−Sca-1+c-Kit::GFP+ cells. Right panels show that after tamoxifen injections GFP expression is initiated in heterozygous c-Kit+/ΔGFP and c-Kit-deficient c-KitW/ΔGFP mice. Data are representative of more than three experiments with three mice per genotype.