Figure 2
Figure 2. Recombination of the 5A3fl interval in vivo and survival of Mx1-Cre, 5A3fl/+ and Mx1-Cre, 5A3fl/fl mice. (A) Analysis of the deleted allele in hematopoietic cells from Mx1-Cre, 5A3fl/+ and Mx1-Cre,5A3fl/fl mice. (Top) PCR analysis of the deleted (del) allele in genomic DNA extracted from peripheral blood from mice of the indicated genotype at the time of killing. Because primers specific for the 5′ and 3′ hprt gene cassettes were used, the 2.3-kb PCR product is obtained only after loxP recombination. To confirm the specificity of the reaction, PCR products were digested with EcoRI, resulting in fragments of 1.5 kb and 0.8 kb. (Bottom) Southern blot analysis of genomic DNA isolated from the bone marrows (BMs) of the same mice as the top panel. The DNA was digested with the Bsu36I restriction enzyme and hybridized with probe B (Figure 1B). The wild-type (wt), floxed (fl), and deleted (del) alleles yield DNA fragments of 9, 15, and 20 kb, respectively. (B) Kaplan-Meier survival curve of wt (+/+; n = 20), 5A3fl/+ (fl/+; n = 20), Mx1-Cre, 5A3fl/+ (fl/+,Cre; n = 35), 5A3fl/fl (fl/fl; n = 22), and Mx1-Cre, 5A3fl/fl (fl/fl, Cre; n = 25) mice (P = .499). Mice that were killed without visible sign of disease were censored. (C) Southern blot analysis of genomic DNA from the bone marrows (BMs) and spleens (SPs) of 5A3fl/+, Mx1-Cre, 5A3fl/fl, and Mx1-Cre, 5A3fl/+ mice 4 days after treatment with a high-dose pIpC regimen. The DNA was digested with Bsu36I and hybridized with probe B as in panel A. (D) Quantitative RT-PCR analysis of the expression of 5 genes within the 5A3 interval in the BM of Mx1-Cre, 5A3+/+ and Mx1-Cre, 5A3fl/+ mice after treatment with high-dose pIpC. Total RNA isolated from the BM of Mx1-Cre, 5A3+/+ (+/+, Cre; n = 3) and Mx1-Cre, 5A3fl/+ mice (fl/+, Cre; n = 3) was analyzed to assess expression of Armc10, Mll5, Napepld, Orc5l, and Psmc2. Expression levels were determined in triplicate, normalized to Gapdh, and calibrated to the levels found in untreated wt mouse bone marrow (+/+; n = 4). The graph shows means ± SD for each group. Statistical significance was determined using the Student t test (*P ≤ .05). (E) Southern blot analysis of BM DNA from a Mx1-Cre, 5A3fl/fl mouse (P) that was killed 4 days after treatment with high-dose pIpC, and its transplant recipient (TP-1) killed 4 months later. TP-2 is the transplant recipient of bone marrow cells from TP-1, and killed 4 months after transplantation. The DNA was digested with Bsu36I and hybridized with probe B as in panel A. A vertical space has been inserted between the TP-1 and TP-2 lanes to indicate that they are from 2 separate experiments.

Recombination of the 5A3fl interval in vivo and survival of Mx1-Cre, 5A3fl/+ and Mx1-Cre, 5A3fl/fl mice. (A) Analysis of the deleted allele in hematopoietic cells from Mx1-Cre, 5A3fl/+ and Mx1-Cre,5A3fl/fl mice. (Top) PCR analysis of the deleted (del) allele in genomic DNA extracted from peripheral blood from mice of the indicated genotype at the time of killing. Because primers specific for the 5′ and 3′ hprt gene cassettes were used, the 2.3-kb PCR product is obtained only after loxP recombination. To confirm the specificity of the reaction, PCR products were digested with EcoRI, resulting in fragments of 1.5 kb and 0.8 kb. (Bottom) Southern blot analysis of genomic DNA isolated from the bone marrows (BMs) of the same mice as the top panel. The DNA was digested with the Bsu36I restriction enzyme and hybridized with probe B (Figure 1B). The wild-type (wt), floxed (fl), and deleted (del) alleles yield DNA fragments of 9, 15, and 20 kb, respectively. (B) Kaplan-Meier survival curve of wt (+/+; n = 20), 5A3fl/+ (fl/+; n = 20), Mx1-Cre, 5A3fl/+ (fl/+,Cre; n = 35), 5A3fl/fl (fl/fl; n = 22), and Mx1-Cre, 5A3fl/fl (fl/fl, Cre; n = 25) mice (P = .499). Mice that were killed without visible sign of disease were censored. (C) Southern blot analysis of genomic DNA from the bone marrows (BMs) and spleens (SPs) of 5A3fl/+, Mx1-Cre, 5A3fl/fl, and Mx1-Cre, 5A3fl/+ mice 4 days after treatment with a high-dose pIpC regimen. The DNA was digested with Bsu36I and hybridized with probe B as in panel A. (D) Quantitative RT-PCR analysis of the expression of 5 genes within the 5A3 interval in the BM of Mx1-Cre, 5A3+/+ and Mx1-Cre, 5A3fl/+ mice after treatment with high-dose pIpC. Total RNA isolated from the BM of Mx1-Cre, 5A3+/+ (+/+, Cre; n = 3) and Mx1-Cre, 5A3fl/+ mice (fl/+, Cre; n = 3) was analyzed to assess expression of Armc10, Mll5, Napepld, Orc5l, and Psmc2. Expression levels were determined in triplicate, normalized to Gapdh, and calibrated to the levels found in untreated wt mouse bone marrow (+/+; n = 4). The graph shows means ± SD for each group. Statistical significance was determined using the Student t test (*P ≤ .05). (E) Southern blot analysis of BM DNA from a Mx1-Cre, 5A3fl/fl mouse (P) that was killed 4 days after treatment with high-dose pIpC, and its transplant recipient (TP-1) killed 4 months later. TP-2 is the transplant recipient of bone marrow cells from TP-1, and killed 4 months after transplantation. The DNA was digested with Bsu36I and hybridized with probe B as in panel A. A vertical space has been inserted between the TP-1 and TP-2 lanes to indicate that they are from 2 separate experiments.

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