Acute postnatal ablation of Hif-2{alpha} results in anemia -- Gruber et al. 104 (7): 2301 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Gruber et al. 10.1073/pnas.0608382104.

Supporting Information

Files in this Data Supplement:

SI Materials and Methods
SI Figure 6
SI Figure 7

SI Figure 6

Fig. 6. The anemia observed in Hif-2aD^/D animals is not due to hemolysis. Blood smears from an Hif-2a^fl/D (A) and an Hif-2aD^/D (B) mouse are normal. (C) Haptoglobin levels in Hif-2aD^/D animals are similar to Hif-2a^fl/D animals.

SI Figure 7

Fig. 7. Serum levels of VEGF are increased 2-fold after PH treatment compared with untreated animals; however, no difference is observed between Hif-2a^fl/D and Hif-2aD^/D animals.

SI Materials and Methods

Generation of the Targeting Vector for the Hif-2a Conditional Allele.
To generate a conditional allele of Hif-2a, a DNA fragment extending 2.5 kbp upstream to 0.5 kbp downstream of coding exon 2 was amplified by PCR (upstream primer: 5'-ACAGGTACCTAAGCACAGAACTTAACCAAATTGGGTCT-3'; downstream primer: 5'-ACATCTAGAGTCAACACAGTGTGGAGCGGGACTCTCG-3'). This fragment was cloned into pCRII (Invitrogen) and a 34-bp loxP site was introduced 0.5 kbp 5' to Exon 2 by site-directed mutagenesis. The modified fragment was then cloned as a KpnI/XbaI fragment into the pLNT vector) in which loxP sites flank the PGK-Neomycin drug resistance cassette (35). A separate DNA fragment extending 7.0 kbp immediately downstream from the Exon 2-containing fragment was amplified by PCR (upstream primer: 5'-CCACTCGAGTGCAGGAAGCCTTTTATCCTTGTCTTCCAG-3'; downstream primer: 5'-ATAGCGGCCGCTAGAATGTTAGGAAGCCCCAGGAGCTAAGT-3') and inserted as a XhoI/NotI fragment into the pLNT vector containing the modified exon 2 fragment. The final targeting vector was linearized with NotI and transfected into RW-4 ES cells. Doubly neomycin- and gancyclovir-resistant ES clones were selected and screened for properly recombined alleles using PCR primers (upstream primer: 5'-AACGTGTTTTCTTCCCCAGATTGAGATG-3'; downstream primer: 5'-GTATGCTATACGAAGTTATAACACTGT-3'). Positive clones were confirmed by Southern analysis of ES cell DNA digested with XbaI and hybridized to a probe as shown in Fig. 1. ES cell clones heterozygous for the properly targeted 3-loxP allele were injected into murine blastocysts and transferred to pseudopregnant females. Germ-line transmission of the targeted allele was confirmed in chimeric offspring by Southern analysis of tail DNAs digested with NcoI. To remove the PGK-Neomycin resistance cassette, generating the final 2-loxP allele, mice heterozygous for the 3-loxP allele were crossed to transgenic E2A-Cre mice, which express Cre protein only very early in embryogenesis (36). We identified mice in the F₁ generation that had lost the PGK-Neomycin cassette but retained exon 2 using multiplex PCR analysis, and crossed them to WT Bl6 mice to segregate the E2A-Cre allele. The resulting Hif-2a 2-loxP allele has loxP sites located 0.5 kbp upstream and downstream of exon 2. Mice homozygous for the 2-loxP allele are phenotypically normal and produce WT Hif-2a mRNA. Cre-mediated recombination between the loxP sites in the 2-loxP allele produces the 1-loxP allele, which lacks exon 2 and produces a mutant mRNA transcript containing multiple in-frame stop codons downstream of exon 1 sequences. The WT, 2-loxP, and 1-loxP allele can be distinguished by a multiplex PCR reaction (primer 1: 5'-CAGGCAGTATGCCTGGCTAATTCCAGTT-3'; primer 2: 5'-CTTCTTCCATCATCTGGGATCTGGGACT-3'; primer 3: 5'-GCTAACACTGTACTGTCTGAAAGAGTAGC-3'). The WT allele produces a 410-bp fragment (primers 1 and 2), the 2-loxP allele a 444 bp fragment (primers 1 and 2), and the 1-loxP allele a 340 bp fragment (primer 2 and 3).

Genotyping for Hif-1a, Ubc-Cre-ER^T2 and ROSAlacZ has been described (26, 27).

All procedures involving mice were performed in accordance with the National Institutes of Health guidelines for use and care of live animals and were approved by the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC).

RNA Preparation.
Total RNA was isolated from cells and organs using TRIzol. cDNA was transcribed from 3 mg of total RNA using Oligo-dT and 18S reverse primers (ABI) and SuperScript first strand synthesis sytem (Invitrogen). PCR primers flanking exon 2 were used to determine full-length mRNA levels transcribed from the recombined locus using the following primers: forward, 5'-ACAATGACAGCTGACAAGGAG-3'; and reverse, 5'-TTGTTGTAGACTCTCACTTGCCC-3' Blood Analysis.
Blood was collected in EDTA-treated tubes (Sarstedt), and blood cell numbers were counted on a Hemavet850 machine. Spun hematocrits were performed on an IEC MicroMB centrifuge. Blood smears on microscope slides were subjected to standard Wright Giemsa staining. CFU Assays.
Bone marrow was flushed out of the femur with PBS; the spleen was macerated in PBS using the plunger of a sterile syringe. Cells were incubated in ACK lysis buffer for 10 min at room temperature to lyse red blood cells, centrifuged for 5 min at 2,000 ´ g, resuspended in IMDM media and filtered through a mesh. Bone marrow cells (10⁶) and 2 ´ 10⁶ spleen cells were plated in 3 ml of methocult M3334 (Epo only) from StemCell Technologies for BFU-E and CFU-E counts, and 10⁵ bone marrow cells were plated in methocult M3434 for all CFUs. Cells were incubated at 37°C, 5% CO₂ and colonies were scored after 7 days. Flow Cytometry.
Bone marrow or spleen cells were isolated as described above. Cells were blocked for 15 min with CD16 antiobdy (1:100 dilution; BD PharMingen), in 0.1% BSA in PBS and stained with PE-conjugated Ter119 (1:100; BD PharMingen) and FITC-conjugated CD71 (1:100; BD PharMingen) antibodies for 30 min and analyzed on a FACSCalibur from BD. Western Blot Analysis.
Western blot analysis was performed as described by using antibodies to HIF-1a (Transduction Labs) and HIF-2a (Novus) (9). Primary antibodies were used at a dilution of 1:500. Secondary antibodies were HRP conjugated, detected by ECL (Amersham Pharmacia), and exposed to autoradiographic film (Kodak). b-Galactosidase Staining.
Organs were fixed in 4% paraformaldehyde at 4°C for 2 h. After 3 washes in PBS at 4°C, organs were incubated in permeabilization solution (2 mM MgCl₂/0.01 % sodium deoxycholate/0.02% Nonidet P-40 in PBS) 3 times for 15 min at room temperature. Organs were stained for 2 h at 37°C in staining solution (2 mg MgCl₂/5 mM potassium ferricyanide/5 mM potassium ferrocyanide/20 mM Tris, pH 7.4/1 mg/ml X-gal/0.02% Nonidet P-40/0.01% deoxycholate in PBS). Staining was stopped by several washes in PBS. Statistical Analysis.
Statistical analysis was performed by using the unpaired Student's t test. P values of <0.05 were considered statistically significant.