Generation of eIF-5A2 transgenic mice
A 462 bp human eIF-5A2 cDNA fragment was cloned into the pCAGGS vector. The linearized constructs were injected into one-cell-stage F1 mouse embryos, which were transplanted into pseudo-pregnant females. All resulting pups were screened for the presence of the transgene using a pair of primers from the vector sequence and a pair of primers from the human eIF-5A2 gene. For studies relying on timed pregnancies, mating pairs were established and mice were monitored daily for vaginal plugs, the presence of which would indicate 0.5 days post-copulation. Animal experimentation was done in accordance with the guidelines of the University of Hong Kong regarding the care and use of laboratory animals.
Western and Northern blot analysis
For Western blot analysis, Protein lysates were prepared with RIPA buffer (1 × PBS, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% SDS and protease inhibitor cocktail). About 10 μg of lysate was separated by SDS-polyacrylamide gel electrophoresis, transferred to a PVDF Hybond-P membrane (Amersham Pharmacia Biotechnology, Piscataway, NJ), and detected by antibodies for eIF-5A2 (a mouse monoclonal antibody raised against the 54 residues of eIF-5A2), p53 (Zymed, San Francisco, CA), p21 (Upstate, Temecula, CA), p19 (Upstate, Lake Placid, NY), CDK4 (Cell Signaling Technology, Beverley, MA), and γ-tubulin (Sigma, St. Louis, MO). For Northern blot analysis, total cellular RNA was prepared using the TRIzol/chloroform method. Twenty microgram of RNA was size fractionated, transferred to a nylon membrane, and hybridized with a 32P-labeled human eIF-5A2 specific probe as described previously[7, 8].
Histological analysis, immunohistochemistry, senescence and BrdU incorporation assay
Fresh mouse tissues were fixed in 4% cold paraformaldehyde in PBS, processed into serial paraffin sections, and stained with Mayer's hematoxylin-eosin staining. Immunohistochemistry (IHC) was performed using the following antibodies: eIF-5A2 (1:500 dilution), PCNA (1:500 dilution, Santa Cruz Biotechnology, Santa Cruz, CA). Internal standardization was achieved by comparing only images stained with the same antibodies in the same experiment, captured with identical parameters, and scaled and displayed identically. For β-galactosidase staining, MEF cells were stained for senescence-associated acidic β-galactosidase activity according to the manufacturer's protocol (Cell Signaling Technology, Beverley, MA). BrdU (100 mg/g of body weight) was injected i.p. into pregnant females. Then the animals were killed 2 h after injection and the mouse tissues or embryos were fixed in 4% paraformaldehyde at 4°C overnight. Then the sections were processed for stained with BrdU staining kit (ZYMED) according to the manufacturer's protocol.
Wound healing experiments
4-month old mice were anaesthetized with methoxyfluorane, and the dorsum was shaved and cleaned with alcohol. Three equidistant 1-cm full-thickness incisional wounds were made through the skin and panniculus carnosus muscle. Wounds were measured at days 1, 2, 3 and 4 post-wounding, and wounded skin specimens were collected and bisected for histology at day 4 post-wounding.
Bone X-ray imaging and calcification analysis
Individual mice were subjected to an X-ray imager for the detection of kyphosis and osteoporosis. In brief, mice were anaesthetized with 2% isofluorane and images were taken by a 600P X-ray mammogram machine (General Electric Co., Albuquerque, NM) with a dose of 15 kV for 100 sec. For calcification analysis, embryos were eviscerated and the skin was removed. The embryos were fixed in 95% ethanol and stained in Alcian blue solution and Alizarin red solution overnight as described previously.
Isolation of mouse embryo fibroblast (MEF) cells
E13.5 embryos were digested at 37°C for 10 min in 0.2% trypsin (Sigma, St. Louis, MO) in PBS (pH 7.4). The cell suspension was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS at 37°C. For cell proliferation analysis, 5 × 104 wild-type and eIF-5A2 transgenic MEF cells were plated on 6-well plates and cell numbers were counted every day for up to 6 days. For flowcytometry test, cells were fixed in 70% ethanol, stained with propidium iodide, and analyzed by flow cytometer.
Cytogenetic analysis, FISH and SKY
Metaphase spreads were prepared from either cultured MEF cells or bone marrow lymphocytes. Metaphases were harvested from cultured MEFs by Colcemid treatment (0.03 μg/ml) for 3 hr or from bone marrow lymphocytes in tested mice by colchincine treatment (3 mg/kg, intraperitoneal injection). Metaphase spreads were stained by standard trypsin-Giemsa banding method and analyzed under microscope as described previously .
For fluorescence in situ hybridization (FISH) analysis, the whole transgene construct was used as a probe, which was labeled with Spectrum Red-dUTP by nick translation (Life Technologies, Inc.). The labeled probe was then hybridized to prebanded wild-type or eIF-5A2 transgenic MEF metaphase chromosomes as described previously .
Spectral karyotyping (SKY) was performed using a SKY probe (Applied Spectral Imaging, Migdal Ha'Emek, Israel) as described previously [19, 20]. The signal detection followed the recommendations of the SKY probe manufacturer. SKY image capturing and karyotyping were performed using the SkyVision Imaging System equipped with a Zeiss Axioplan 2 fluorescence microscope.
Telomerase activity assay
The telomere repeat amplification protocol (TRAP) assay was performed using the TRAPEZE kit (Invitrogen, New York, NY) following the manufacturer's protocol. Except for negative control (lysis buffer), 0.05 μg protein was used for each PCR, which was run for 33 cycles.
Statistical analysis was performed using the SPSS software (SPSS Standard version 8.0). Significance of difference was analyzed using Student's t tests. A significant difference was considered when the p value was less than 0.05.