![Figure 1. Generation of Gfi1b:GFP knock-in mice. (A) Schematic representation of the targeting construct design and simplified restriction maps of the wt and targeted alleles after CRE recombinase excision of the thymidine kinase-neomycin cassette (TK-neo) by crossing of heterozygous mice with CRE-transgenic mice. All coding sequences of Gfi1b are replaced by the GFP open reading frame. lxP indicates lox-P site; H, HindIII; E, EcoRI; N, NotI; Sl, SalI; B, BamHI; DTA, diphtheria toxin; 1-7, exon 1 to 7; GFP, green fluorescent protein. (B) Southern blotting to detect excision of the thymidine kinase-neomycin resistance gene fusion protein (TK-neo) cassette in mice after gene targeting and CRE-recombination showed the expected 8-kb fragment using a 5′-external probe. (C) PCR strategy and results to detect the insertion of the GFP cDNA into the Gfi1b locus in mouse embryo tail DNA representing the embryos shown in panel D. Because all 5′ sequences are retained, but intron 2 to intron 6 were deleted in the targeted allele, the wt, wt/KI (Gfi1b+/GFP), and KI/KI (Gfi1bGFP/GFP) mice were analyzed by multiplex PCR covering either intron 1 to 2 or intron 1 to GFP (Figure 1C) to demonstrate proper targeting. (D) Appearance of (top panel) and detection of GFP fluorescence in day 13.5 postconception (pc) embryos which were wt, heterozygous, or homozygous for the GFI1b:GFP allele (wt, wt/KI [Gfi1b+/GFP] and KI/KI [Gfi1bGFP/GFP], bottom panel). In heterozygous embryos, GFP expression is restricted to the fetal liver, where erythropoiesis takes place. (Bottom panel) CD31 whole-mount staining showing normal vascularization in wt and mutant embryos. Embryos were analyzed with the Leica MZ/FLIII stereomicroscope (Leica, Wetzlar, Germany) and photographed with the KAPPA CF 15/4 MC camera (KAPPA Opto-Electronics, Gleichen, Germany) using 0.5× magnification (0.125 NA). (E) Western blot analysis of whole-cell extracts from fetal liver cells of wt or homozygous GFP knock-in embryos at day 13.5 pc showing the loss of Gfi1b expression (top) and gain of GFP expression (bottom) in Gfi1b:GFP knock-in mice.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/109/6/10.1182_blood-2006-06-030031/4/zh80060709530001.jpeg?Expires=1723232259&Signature=B8-QvQvOEycJwVaWQi~1qBZ1kSJQtzt9uZC55lt47Xrd~83e1C~P7DLa02R-evpBZut8GJXpbZKjRxOLllb7TJqUuTurlFlnec4pj~GfhKE0wslbT~sugbylhyt3lQHW28hFS2BUAVzzJQOUEBzfxx5JLvW61owj9MnKfa8Oc0s~oO0zjR0cxaoVMcHY37obHrFn8qDuk7o08DMNbeJB0xHx6K1s84vx1v5vS0CnFvlQPfyshtY7wuFDMebJWS2vhk5FL-vQyLzvDWUCOROExEk80NoO5wdF3DmvT2aHJRKOmgKE4bQPpd5n7JhUG8OJZaCMDoCTbneZJdmSimHyKw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Generation of Gfi1b:GFP knock-in mice. (A) Schematic representation of the targeting construct design and simplified restriction maps of the wt and targeted alleles after CRE recombinase excision of the thymidine kinase-neomycin cassette (TK-neo) by crossing of heterozygous mice with CRE-transgenic mice. All coding sequences of Gfi1b are replaced by the GFP open reading frame. lxP indicates lox-P site; H, HindIII; E, EcoRI; N, NotI; Sl, SalI; B, BamHI; DTA, diphtheria toxin; 1-7, exon 1 to 7; GFP, green fluorescent protein. (B) Southern blotting to detect excision of the thymidine kinase-neomycin resistance gene fusion protein (TK-neo) cassette in mice after gene targeting and CRE-recombination showed the expected 8-kb fragment using a 5′-external probe. (C) PCR strategy and results to detect the insertion of the GFP cDNA into the Gfi1b locus in mouse embryo tail DNA representing the embryos shown in panel D. Because all 5′ sequences are retained, but intron 2 to intron 6 were deleted in the targeted allele, the wt, wt/KI (Gfi1b+/GFP), and KI/KI (Gfi1bGFP/GFP) mice were analyzed by multiplex PCR covering either intron 1 to 2 or intron 1 to GFP (Figure 1C) to demonstrate proper targeting. (D) Appearance of (top panel) and detection of GFP fluorescence in day 13.5 postconception (pc) embryos which were wt, heterozygous, or homozygous for the GFI1b:GFP allele (wt, wt/KI [Gfi1b+/GFP] and KI/KI [Gfi1bGFP/GFP], bottom panel). In heterozygous embryos, GFP expression is restricted to the fetal liver, where erythropoiesis takes place. (Bottom panel) CD31 whole-mount staining showing normal vascularization in wt and mutant embryos. Embryos were analyzed with the Leica MZ/FLIII stereomicroscope (Leica, Wetzlar, Germany) and photographed with the KAPPA CF 15/4 MC camera (KAPPA Opto-Electronics, Gleichen, Germany) using 0.5× magnification (0.125 NA). (E) Western blot analysis of whole-cell extracts from fetal liver cells of wt or homozygous GFP knock-in embryos at day 13.5 pc showing the loss of Gfi1b expression (top) and gain of GFP expression (bottom) in Gfi1b:GFP knock-in mice.
