Figure 4
Figure 4. Conditional Ubap2l deletion results in loss of HSC activity. (A) Targeting strategy to generate the conditional and knockout Ubap2l mice. 2-5, Ubap2l exons; LoxP, Cre-recombinase recognition site; Frt, Flpe recombinase recognition site; Neo, neomycin selection cassette. (B) Southern blot analysis of genomic DNA isolated from selected ES clones to confirm the proper targeting of the Ubap2l locus. The introduction of an extra EcoRI site located immediately before the 5′ LoxP site allowed for the detection of an additional genomic restriction fragment of 3.59 kb when using radiolabeled probe hybridizing to a region located upstream of the targeted region (left panel). The correct recombination of the targeted region was verified at the 3′ end by the detection of a 8.58-kb fragment corresponding to the insertion of the FRT-pgk-NEO-pA-FRT-LoxP cassette using a probe hybridizing downstream of the targeted region (right panel). Single integration of the targeting fragment was confirmed by the detection of the same band using a NEO probe (data not shown). Efficient recombination between the LoxP and FRT sites was monitored in ES clones after transfection with either Flpe- or Cre-expressing plasmid by the detection of restriction fragments of the appropriate length using the indicated probes. (C) Relative Ubap2l mRNA expression in MEFs. 3-4, 9-10, 14-15, and 21-22 refer to the exons targeted by the primers used for qRT-PCR. The values are expressed relative to Hprt. The average of 3 independent experiments is presented with the standard deviation. The values for MEFs +/+ were set to 1. (D) Western blot validating the absence of UBAP2L protein in Ubap2l−/− MEFs. (E) Competitive repopulation activity of conditional Ubap2l knockout BM in recipient’s peripheral blood at different time points (4 weeks posttransplantation and 18 days, 4 weeks, 8 weeks, 12 weeks, 16 weeks, and 20 weeks post-pIpC treatment). Shown are means with standard deviation (n = 15 mice per genotype). The Mann-Whitney test was used for statistical analysis. *P < .05. (F) Competitive repopulation activity of conditional Ubap2l knockout BM in recipient’s BM 28 weeks post-pIpC treatment. Shown are means with standard deviation (n = 6 mice per genotype). The Mann-Whitney test was used for statistical analysis. *P < .05.

Conditional Ubap2l deletion results in loss of HSC activity. (A) Targeting strategy to generate the conditional and knockout Ubap2l mice. 2-5, Ubap2l exons; LoxP, Cre-recombinase recognition site; Frt, Flpe recombinase recognition site; Neo, neomycin selection cassette. (B) Southern blot analysis of genomic DNA isolated from selected ES clones to confirm the proper targeting of the Ubap2l locus. The introduction of an extra EcoRI site located immediately before the 5′ LoxP site allowed for the detection of an additional genomic restriction fragment of 3.59 kb when using radiolabeled probe hybridizing to a region located upstream of the targeted region (left panel). The correct recombination of the targeted region was verified at the 3′ end by the detection of a 8.58-kb fragment corresponding to the insertion of the FRT-pgk-NEO-pA-FRT-LoxP cassette using a probe hybridizing downstream of the targeted region (right panel). Single integration of the targeting fragment was confirmed by the detection of the same band using a NEO probe (data not shown). Efficient recombination between the LoxP and FRT sites was monitored in ES clones after transfection with either Flpe- or Cre-expressing plasmid by the detection of restriction fragments of the appropriate length using the indicated probes. (C) Relative Ubap2l mRNA expression in MEFs. 3-4, 9-10, 14-15, and 21-22 refer to the exons targeted by the primers used for qRT-PCR. The values are expressed relative to Hprt. The average of 3 independent experiments is presented with the standard deviation. The values for MEFs +/+ were set to 1. (D) Western blot validating the absence of UBAP2L protein in Ubap2l−/− MEFs. (E) Competitive repopulation activity of conditional Ubap2l knockout BM in recipient’s peripheral blood at different time points (4 weeks posttransplantation and 18 days, 4 weeks, 8 weeks, 12 weeks, 16 weeks, and 20 weeks post-pIpC treatment). Shown are means with standard deviation (n = 15 mice per genotype). The Mann-Whitney test was used for statistical analysis. *P < .05. (F) Competitive repopulation activity of conditional Ubap2l knockout BM in recipient’s BM 28 weeks post-pIpC treatment. Shown are means with standard deviation (n = 6 mice per genotype). The Mann-Whitney test was used for statistical analysis. *P < .05.

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