Figure 1
Figure 1. Generation of gimap5 knockout mice. (A) A replacement-type gene-targeting vector was constructed from a thymidine kinase expression cassette (TK); a 5.4-kb EcoR1-BamH1 fragment (5′-homology) of gimap5 intron 1; a pkg-promoter-neomycin resistance gene cassette (NEOR, orientation opposite to gimap5) and a 1.3-kb fragment comprising the last 713 bp of exon 3 plus 597 bp of 3′-flanking region. Homologous recombination replaces a 2466-bp fragment of the Gimap5 gene containing exon 2 and a fragment of exon 3, thereby deleting the translation start signal within exon 2 and the first 130 amino acids of gimap5 protein. The figure is not drawn to scale. (B) Recombination in the 5′ and 3′ region of the Gimap5 gene was confirmed by amplification of a 4-kb genomic fragment with primers A and B (ii), and a 2.2-kb fragment with primers C and D (iii), respectively. * indicates ES clones having undergone the predicted recombination events in both the 5′ and 3′ homology. (iv) Southern blot hybridization of Xba1-digested genomic DNA from Gimap+/− ES clones (lanes 1,2) or wild-type ES cells (lane 3) with a 32P-labeled neomycin probe detects the predicted 3.1-kb fragment, consistent with a single locus integration of the NEO cassette. (v) Southern blot hybridization analysis of Bgl2-digested genomic DNA from wild-type (lane 1) and Gimap5−/− mice (lane 2) with probe E. The probe detects in wild-type mice a 2.6-kb fragment comprising exon 3 of gimap5 and a 1.8-kb fragment containing a homologous region in exon 5 of Gimap3; in Gimap5−/− DNA, the Gimap3 fragment detected is identical to wild-type, whereas the Gimap5 fragment shifts to 4.1 kb resulting from insertion of the NEO gene. (C) Measurements of gimap gene family mRNA abundance by real-time PCR. The structure of the gimap gene cluster on chromosome 6 is shown on top. Bars indicate the average plus or minus SEM from triplicate determinations from 3 mice each. Data are normalized for gimap abundance in wild-type C57BL/6J mice. Only gimap5 mRNA is significantly reduced in Gimap5+/− mice and is undetectable in Gimap5−/− mice. (D) Survival of Gimap5−/− mice (n = 35) over 42 weeks. The median age of death lies between 14 and 15 weeks. By 42 weeks, no surviving Gimap5−/− mice remained.

Generation of gimap5 knockout mice. (A) A replacement-type gene-targeting vector was constructed from a thymidine kinase expression cassette (TK); a 5.4-kb EcoR1-BamH1 fragment (5′-homology) of gimap5 intron 1; a pkg-promoter-neomycin resistance gene cassette (NEOR, orientation opposite to gimap5) and a 1.3-kb fragment comprising the last 713 bp of exon 3 plus 597 bp of 3′-flanking region. Homologous recombination replaces a 2466-bp fragment of the Gimap5 gene containing exon 2 and a fragment of exon 3, thereby deleting the translation start signal within exon 2 and the first 130 amino acids of gimap5 protein. The figure is not drawn to scale. (B) Recombination in the 5′ and 3′ region of the Gimap5 gene was confirmed by amplification of a 4-kb genomic fragment with primers A and B (ii), and a 2.2-kb fragment with primers C and D (iii), respectively. * indicates ES clones having undergone the predicted recombination events in both the 5′ and 3′ homology. (iv) Southern blot hybridization of Xba1-digested genomic DNA from Gimap+/− ES clones (lanes 1,2) or wild-type ES cells (lane 3) with a 32P-labeled neomycin probe detects the predicted 3.1-kb fragment, consistent with a single locus integration of the NEO cassette. (v) Southern blot hybridization analysis of Bgl2-digested genomic DNA from wild-type (lane 1) and Gimap5−/− mice (lane 2) with probe E. The probe detects in wild-type mice a 2.6-kb fragment comprising exon 3 of gimap5 and a 1.8-kb fragment containing a homologous region in exon 5 of Gimap3; in Gimap5−/− DNA, the Gimap3 fragment detected is identical to wild-type, whereas the Gimap5 fragment shifts to 4.1 kb resulting from insertion of the NEO gene. (C) Measurements of gimap gene family mRNA abundance by real-time PCR. The structure of the gimap gene cluster on chromosome 6 is shown on top. Bars indicate the average plus or minus SEM from triplicate determinations from 3 mice each. Data are normalized for gimap abundance in wild-type C57BL/6J mice. Only gimap5 mRNA is significantly reduced in Gimap5+/− mice and is undetectable in Gimap5−/− mice. (D) Survival of Gimap5−/− mice (n = 35) over 42 weeks. The median age of death lies between 14 and 15 weeks. By 42 weeks, no surviving Gimap5−/− mice remained.

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