Fig. 1.
Fig. 1. Creating an Hp null mouse. (A) Targeting strategy. Solid boxes denote exons 1 to 5. The vector in a 5′ to 3′ direction consists of a 3.1-kb EcoRI/Pst I fragment that includes sequences upstream of the hp gene, exon I and part of intron 1, a neomycin-resistant gene, a 1.3-kbBamHI fragment that includes part of intron 4 and exon 5, and a Herpes simplex viral thymidine kinase gene. Exons 2, 3, and 4 were deleted in a targeted allele. A 1.8-kbBamHI/HindIII fragment that includes part of exon 5 and the 3′ flanking sequences and lies outside the region of homology was identified as a single-copy probe and used in Southern blot analysis. (B) Southern blot analysis of progenies from Hp+/− inter-crosses using EcoRI digestion and the 1.8-kbBamHI/HindIII fragment as probe. The 7.1-kb fragment represents the wild-type allele and the 3.5-kb fragment represents the targeted allele. (C) Verification of Hp null phenotype by RT-PCR. RT-PCR using RNA from livers of +/+, +/−, and −/− mice at 0, 6, and 24 hours after LPS injection of 0.1 mg/10 g body weight. Briefly, 2 μg RNA from each genotype at various time points was reverse transcribed to cDNAs. The cDNAs were diluted 1× and 10× and amplified by PCR in the presence of 32P-dCTP usingHaptoglobin (hp) and triose phosphate isomerase (TPI) -specific primers to give 895- and 523-bp fragments, respectively. The RT-PCR products were separated on a 5% polyacrylamide gel, quantitated by phosphorimaging, and exposed to autoradiography. The Hpsignal was normalized against the TPI signal. No Hp mRNA was observed for −/− mice. There was about twice as much Hp mRNA in the liver of +/+ mice as there is in the +/− mice, and this ratio was maintained during induction by LPS. (D) Verification ofHp null phenotype by Western blot analysis. Mice were treated with LPS and 1 μL of plasma taken from each mouse 24 hours after treatment time was analyzed by Western blot analysis as described in Materials and Methods. There was no detectable Hp for −/− mice. Lane H represents the human Hp standard. (E) Genotype distribution of 3-week-old progenies from 10 different heterozygous breeder pairs and 18.5 dpc embryos from 13 different heterozygous crosses. For quantitation of plasma Hb, 5- to 6-week-old mice were lightly anesthetized and blood from the tail vein was collected in a heparinized capillary tube. The mean plasma Hb level in −/− mice was not significantly different from that in +/+ mice (P= .21).

Creating an Hp null mouse. (A) Targeting strategy. Solid boxes denote exons 1 to 5. The vector in a 5′ to 3′ direction consists of a 3.1-kb EcoRI/Pst I fragment that includes sequences upstream of the hp gene, exon I and part of intron 1, a neomycin-resistant gene, a 1.3-kbBamHI fragment that includes part of intron 4 and exon 5, and a Herpes simplex viral thymidine kinase gene. Exons 2, 3, and 4 were deleted in a targeted allele. A 1.8-kbBamHI/HindIII fragment that includes part of exon 5 and the 3′ flanking sequences and lies outside the region of homology was identified as a single-copy probe and used in Southern blot analysis. (B) Southern blot analysis of progenies from Hp+/− inter-crosses using EcoRI digestion and the 1.8-kbBamHI/HindIII fragment as probe. The 7.1-kb fragment represents the wild-type allele and the 3.5-kb fragment represents the targeted allele. (C) Verification of Hp null phenotype by RT-PCR. RT-PCR using RNA from livers of +/+, +/−, and −/− mice at 0, 6, and 24 hours after LPS injection of 0.1 mg/10 g body weight. Briefly, 2 μg RNA from each genotype at various time points was reverse transcribed to cDNAs. The cDNAs were diluted 1× and 10× and amplified by PCR in the presence of 32P-dCTP usingHaptoglobin (hp) and triose phosphate isomerase (TPI) -specific primers to give 895- and 523-bp fragments, respectively. The RT-PCR products were separated on a 5% polyacrylamide gel, quantitated by phosphorimaging, and exposed to autoradiography. The Hpsignal was normalized against the TPI signal. No Hp mRNA was observed for −/− mice. There was about twice as much Hp mRNA in the liver of +/+ mice as there is in the +/− mice, and this ratio was maintained during induction by LPS. (D) Verification ofHp null phenotype by Western blot analysis. Mice were treated with LPS and 1 μL of plasma taken from each mouse 24 hours after treatment time was analyzed by Western blot analysis as described in Materials and Methods. There was no detectable Hp for −/− mice. Lane H represents the human Hp standard. (E) Genotype distribution of 3-week-old progenies from 10 different heterozygous breeder pairs and 18.5 dpc embryos from 13 different heterozygous crosses. For quantitation of plasma Hb, 5- to 6-week-old mice were lightly anesthetized and blood from the tail vein was collected in a heparinized capillary tube. The mean plasma Hb level in −/− mice was not significantly different from that in +/+ mice (P= .21).

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