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Stoffel et al. 10.1073/pnas.0406380102. |
Supporting Text
Generation of the smpd3-/- and smpd2-/- ´smpd3-/- Double Mutant Mouse Line. A 15-kb genomic SstI fragment covering the entire SMPD3 coding region was isolated from a mouse genomic l-phage clone (129/SvEvTacfBR), cloned into pBluescript II SK+ and characterized by restriction enzyme mapping using murine smpd3 cDNA probes. EcoRI digestion yielded a 8.4-kb EcoRI fragment, of which a 2-kb KpnI-BamHI fragment with the 1.4-kb exon I was inserted into the BamHI/KpnI restricted pcDNA3.1/Myc-His and cloned as a XhoI 5' fragment into the XhoI restricted pPNT vector. A 4.4-kb BamHI fragment of the 8.4-kb fragment was inserted into the BamHI site of this pPNT as a 3' fragment. The orientation of both inserts was determined by DNA sequencing and restriction analysis (Fig. 1). The NotI restricted targeting construct was electroporated into CJ7 ES cells. Clone selection, genotyping by Southern blotting and PCR, C57BL/6 blastocyst injection, and selection of germline chimeric males and their crossing to smpd3-/- were carried out following established protocols (1) and as described (2).
Southern blot analysis of SstI-digested genomic DNA of correctly targeted ES clones and tail DNA of chimeric mice yielded a 6.4-kb fragment of the smpd2 locus and a 4.8-kb fragment of the wt allele, when probed with the 1.2-kb XhoI-EcoRI fragment as external probe (Fig. 1C). PCR with primers NSM2 ATG s 5'-ATG GTT TTG TAC ACG ACC CCC TTT CCT AAT-3' and NSM2 ExIas 5'-CCT GAG AAA CAG AGC TCC CTT AGA GGC CAG-3' confirmed the homologous recombination.
smpd2-/-smpd3-/- double null mice were generated by crossing smpd2-/- female mice and smpd3-/- males. The smpd2-/- locus was probed with the EcoRI-XbaI restriction fragment length polymorphisms (RFLPs) (WT, 8.5 kb; -/-, 6 kb) in Southern blot analysis and by PCR by using primer pair Xhosense (5'-CTCGAGTTGCTCCGAAGCACTCCAGCCATG-3') and RT3' as (5'-GTGTGTCAAGCAAGTTTTATTGTTAAGCTC-3') for probing the smpd2 locus. The wt yielded a 3.2-kb and the smpd2-/- and NSM2 ExIas yielded a 1.3-kb fragment for wt and a 3.1-kb fragment for smpd3-/- mutant DNA (2).
Expression Studies Followed by Semiquantitative RT-PCR (3). Total RNA was extracted from pituitaries of p20 wt and scd1-/- mice by the TRIzol method following the manufacturer’s instruction (GIBCO/BRL). Contaminating genomic DNA was removed by DNase I treatment (25 units/10µg of total RNA). cDNA was synthesized by using Maloney murine leukemia virus reverse transcriptase, random hexamer primers, and dNTPs. cDNA templates were amplified by PCR with specific primers, listed below, in the presence of dNTPs, [a -32P] CTP, and Taq polymerase. Quantitative PCR amplification was optimized for each primer pair at 15, 20, 25, and 30 cycles to ensure the linear range and carried out in the presence of 1 µCi [a -32P] dCTP.
The following oligonucleotide primers were used for RT-PCR: mGHRHR s ACC CAT AGT CCT CTC TGT TGG GGT GAA CTT; mGHRHR as GTT CAC CCC AAC AGA GAG GAC TAT GGG TCC; mGHRH s AAG GAT GCT GCT CTG GGT GCT CTT TGT GAT; mGHRH as GGG GCT CGG GGG GCT CAA GCG TCC GCT GAA; pit 1 s AAT GCT TGT AAA CTC AAA GCA ATT TTA TCC; pit 1 as TCT GCA CTC TAG ATG GTC CTT GGA AAT AGA; prop1 s GGC TAG CCA TGG AAG CTC AAA GGA GCC; prop1 as AAA AGT TAG TTC CAG GAC TTT GGC GTC TCA
Hormone Assays. Serum FT3-, FT4-, and cortisol assays were kindly carried out on a ACS:Centaur by G. Assmann (Institute of Clinical Chemistry, University of Münster, Münster, Germany). Pooled sera of cohorts of five p20 male and female wt and smpd3-/- siblings and five adult (3 mo) smpd3-/- and control male mice used for the assays.
Immunocytochemistry. Pituitary hormone antibodies were kindly supplied by A. F. Parlow (National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Disease, Torrance, CA) and anti-GH-releasing hormone (GHRH) by G. Thordarson (University of California, Santa Cruz).
Subcellular Fractionation and Protein Analysis. Brain tissue and cultured cells were homogenized in homogenization buffer (50 mM Tris, pH 7.4/10 mM EDTA/10 mM EGTA/5 mM DTT/0.32 M sucrose/2´
Complete without EDTA). The membrane fraction was sedimented from the 2,000 ´
g supernatant at 20,000 ´
g for 15 min and fractionated in a discontinuous sucrose gradient (2, 1.6, 1.4, 1.2, and 0.8 M sucrose in 10 mM Tris, pH 7.4) at 100,000 ´
g for 2 h in a SW41 Beckman rotor. One-milliliter fractions were collected from bottom to top for protein analysis and enzyme assay. The Golgi fraction floated at the 1.2-0.8 M sucrose layer (density 1.104 g/ml). Triton X-100 insoluble membrane domains (DIMs) were isolated from the Golgi fraction as described (4). The DIM fractions (1-3) with densities 1.030-1.075 g/ml were pooled. Protein was determined by Bradford assay (5).
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2. Zumbansen, M. & Stoffel, W. (2002) Mol. Cell. Biol. 22, 3633-3638.
3. Wilson, P. A. & Melton, D. A. (1994) Curr. Biol. 4, 676-686.
4. Simons, M., Kramer, E. M., Thiele, C., Stoffel, W. & Trotter, J. (2000) J. Cell. Biol. 151, 143-154.
5. Bradford, M. M. (1976) Ann. Biochem. 72, 248-254.