Mice carrying a disrupted La allele.
Mice carrying a disrupted La allele were generated by Ingenious Targeting Laboratory (Stony Brook, NY). The targeting vector contained 9.5 kb of genomic DNA upstream of the mouse La exon 1, followed by a neomycin resistance cassette plus 900 bp from intron 2 (see Fig. ). The neomycin resistance gene (driven by the phosphoglycerate kinase I promoter in the orientation opposite that of La) replaced La gene exons 1 and 2, the latter of which contained the ATG start codon. NotI-linearized targeting vector was transfected into 129SV/EV embryonic stem cells (Ingenious Targeting Laboratory, Stony Brook, NY). Clones were selected and expanded in G418, and PCR analysis identified ones that had undergone homologous recombination, which were microinjected into C57BL/6J and BALA/cj blastocysts. The resulting chimeric mice were mated with C57BL/6 mice, and those that exhibited germ line transmission of the disrupted La gene were used to produce a founding pair of La+/− mice (LaKO21 and LaKO27), both of which obtained the disrupted mLa allele from the same targeted ES cell clone.
FIG. 2. Creation of a disrupted mLa gene. (A) Schematic showing the structures of the wild-type genomic mLa locus, targeting vector, and disrupted allele. The first 4 of the 12 exons of the mouse La gene are shown. Exon 2 (Ex2) contains the ATG and encodes the (more ...) Genotyping.
DNA was purified from tail clippings, typically at 3 weeks of age, using a DNeasy tissue kit (catalog no. 69506; Promega), examined, and quantitated by gel electrophoresis and ethidium staining. Triplex PCR containing 200 ng of DNA and three primers was used to assay for disrupted and wild-type mLa alleles simultaneously; allele-specific primers (wild type [5′-ATAGGCCACAATGGCTGAAAATGGAG] and disrupted [5′-TTATGGGCAATTTCCCCACAGCCA]) together with a common reverse primer (5′-TGCGAGGCCACAGGCCACTT) were used. PCR parameters were as follows: (i) 94°C for 1 min; and (ii) 30 cycles, with 1 cycle consisting of 94°C for 45 s, 67°C for 25 s, and 68°C for 45 s. The expected products from wild-type and disrupted alleles are 700 and 500 bp long, respectively. Genotypes of all mice used for breeding, embryo production, and Southern and Northern blotting were confirmed by two additional tail clips and genotyping.
Preimplantation embryos were flushed from uteri, and individual embryos were incubated overnight at 55°C in 15 μl of embryo lysis buffer (50 mM KCl, 10 mM Tris-HCl, pH 8, 2 mM MgCl2, 0.45% Nonidet P-40 [NP-40], 0.45% Tween 20, and 0.1 mg/ml proteinase K). The proteinase was then inactivated by incubation at 100°C for 2 min, and 8-μl samples of the lysed embryos were added to 17 μl of PCR master mix containing 10× PCR buffer, primer, deoxynucleoside triphosphates, and Taq polymerase (BD Biosciences). PCR mixtures were heated at 94°C for 1 min and then subjected to 35 cycles, with 1 cycle consisting of 94°C for 45 s and 68°C for 2 min.
Southern and Northern blotting.
DNA obtained from the livers of La+/+ and La+/− mice was digested with AflIII, EcoRI, or HindIII, separated on 0.9% agarose gel, denatured, and transferred to a nylon membrane (GeneScreen Plus; Perkin-Elmer Life Sciences). Probes for Southern analysis were derived from mLa gene intron 2 and the Neo gene as indicated in Fig. . Probes were labeled by random priming with [α-32P]dCTP (Lofstrand Labs, Gaithersburg, MD). Hybridization was in QuikHyb solution processed accordingly (Stratagene). The membrane was exposed to a Fuji phosphorimager screen and analyzed using Image Gauge software.
Adult mouse multiple tissue and mouse embryo Northern blots were obtained from Clontech. Full-length mLa and beta-actin cDNAs were labeled by random priming with [α-32P]dCTP. For La+/+ and La+/− samples, total RNA was prepared from tissue dissected from adult littermates using TRIzol (Invitrogen), and 30 μg was electrophoresed and transferred to a charged nylon membrane (GeneScreen Plus; Perkin-Elmer Life Sciences, Inc.). Blots were probed first with a radiolabeled 0.7-kb 3′ fragment of mLa cDNA followed by beta-actin cDNA. Hybridization was performed as described above for Southern blotting.
Cloning, expression, and purification of His-tagged recombinant mLa antigen and affinity purification of anti-mLa Ab.
mLa cDNA (30
) was modified to encode a C-terminal His6
tag and cloned into the NcoI-XhoI fragment of pET-28a (Novagen). After 4 h of induction by isopropyl-β-d
-thiogalactopyranoside (IPTG), purification was performed by nickel-agarose chromatography (QIAGEN). Anti-mLa serum was obtained from a rabbit immunized with purified recombinant mLa. For affinity purification, a preparative sodium dodecyl sulfate gel containing purified recombinant mLa was electroblotted onto a nitrocellulose strip, and the band corresponding to full-length mLa was cut out and used as an affinity matrix (25
). Briefly, the nitrocellulose strips were washed with 100 mM glycine-HCl, pH 2.5, and then with phosphate-buffered saline (PBS) containing 0.05% NP-40, blocked with 5% bovine serum albumin in PBS, and incubated with anti-mLa serum for 3 h at room temperature. The strips were washed, eluted with 100 mM glycine-HCl (pH 2.5), and neutralized with Tris base to pH 7.5. The concentration of the affinity-purified antibody (Ab) was determined by comparison to the concentration of purified rabbit immunoglobulin G (IgG) (Zymed Labs, Inc.) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining.
Liver and 32D cells were washed once with ice-cold PBS containing 1 mM phenylmethanesulfonyl fluoride (PMSF), and lysed in ice-cold lysis buffer containing 1% NP-40, 0.5 M NaCl, 10 mM HEPES, pH 7.5, 10% glycerol, 1 mM PMSF, and complete protease inhibitor cocktail (Roche). The liver was homogenized on ice with a Brinkmann polytron (Kinematica) and sonicated on ice with an XL-2020 sonicator (Misonix) equipped with a Misonix microtip probe 419 (3.2-mm tip diameter; 16.5-cm length) at 550 W; the liver was sonicated three times for 10 s each time. The samples were then rocked gently for 30 min at 4°C and cleared by centrifugation at 15,000 × g for 10 min. Extraction of 32D cells was the same except without polytron homogenization. Protein quantitation was performed by using Bio-Rad protein reagent. Cleared lysates (50 μg) were separated on a precast 12% Tris-glycine polyacrylamide gel (Invitrogen) and transferred to a nitrocellulose membrane. The membrane was incubated with anti-mLa serum or affinity-purified anti-mLa and visualized with 125I-protein A (Amersham Biosciences).
Blastocyst outgrowth, immunofluorescence (IF), and genotyping.
Blastocysts were recovered at 3.5 days postcoitus and cultured individually in Dulbecco's modified Eagle's medium (Gibco catalog no. 10313-021) supplemented with 15% ES cell-certified fetal bovine serum, 15 mM HEPES buffer, 100 units/ml of penicillin, 100 μg/ml of streptomycin, 100 μM nonessential amino acids, 4.5 mM of l-glutamine, and 100 μM of β-mercaptoethanol on gelatinized chamber slides (Nalge Nunc International) at 37°C in 5% CO2. The blastocysts were visualized using an Olympus LH50A microscope equipped with a Polaroid DMC Ie digital camera. The chamber slides were incubated in 4% paraformaldehyde in PBS for 30 min and washed in PBS, and the embryos were permeabilized and fixed with 100% methanol for 20 min, washed, and blocked with PBS containing 3% bovine serum albumin and 0.1% Tween 20 for 30 min. The embryos were then washed and incubated with TROMA-I antibody (Developmental Studies Hybridoma Bank) at 1:25 in the blocking solution overnight at 4°C. The embryos were washed and incubated with Texas red-conjugated goat anti-rat IgG (1:200 in blocking solution; Vector Laboratories). The embryos were then washed, incubated in 0.5 μg/ml of purified anti-mLa antibody in blocking solution overnight at 4°C, washed, incubated with FITC-conjugated goat anti-rabbit IgG (1:200 in blocking solution), and washed. The embryos were then mounted on slides in SlowFade Light Antifade medium with 4′,6′-diamidino-2-phenylindole (DAPI) (1.5 μg/ml; Molecular Probes) and observed with a Nikon Eclipse E600 microscope equipped with a Nikon digital camera DXM1200F, using ACT-1 and Adobe software. For genotyping, individual embryos were recovered from the slides, transferred to PCR tubes, and processed as described above.
ES cell lines.
Embryonic stage E3.5 (embryonic day 3.5) blastocysts were isolated and cultured on mitomycin C-treated mouse embryonic fibroblasts in Dulbecco's modified Eagle's medium (Gibco catalog no. 10313-021) for 4 days as described previously (24
). The inner cell mass (ICM) was picked, trypsinized, replated, and cultured for 7 days. Undifferentiated ES cell colonies were picked, trypsinized, and expanded.