Exon trapping was performed as described earlier (19
). Briefly, a bacterial artificial chromosome clone encompassing 150 kb of the Evi12
locus was partially digested with HpaII. Fragments were cloned into the exon trap vector pERVF0, pooled, and transfected into COS cells. RNA was isolated after 2 or 3 days and used to amplify potential exons by RT-PCR. Southern blot analysis confirmed the presence of one of the isolated potential exons (Gnn
exon 3) on a 2.8-kb EcoRI/BamHI genomic fragment near Evi12
Synthesis of cDNA.
Total RNA was extracted from cell lines and adult organs using guanidinium isothiocyanate or Trizol (Gibco BRL, Breda, The Netherlands). Reverse transcriptase reactions were performed with 5 μg of RNA using random hexamers, oligo(dT), or oligo(dT) adapter primer [5′-GTCGCGAATTCGTCGACGCG(dT)15-3′] for 3′ rapid amplification of cDNA ends (3′ RACE) using the superscript cDNA amplification kit (Gibco BRL).
Identification of Gnn sequences.
A mouse 17-day Embryo MATCHMAKER (MM) cDNA library (BD Biosciences, Palo Alto, Calif.) was initially used to amplify additional Gnn sequences. The locations of amplified Gnn cDNA fragments cDNA1 to cDNA4 are indicated in Fig. . The sequences of cDNA1 were amplified by PCR using primers for exon 3: 5′ sequences were amplified using pACT2MM-5′ (BD Biosciences) and 5′-ACCTCCTCATGGTCTGTGGG-3′ and then pACT2MM-5′ and 5′-GGTAAAAGGGCCATATCTTC-3′, and 3′ sequences were amplified using pACT2MM-3′ (BD Biosciences) and 5′-GAAGATATGGCCCTTTTACCC-3′ and then pACT2MM-3′ and 5′-CACAGACCATGAGGAGGT-3′. The full-length coding region of cDNA1 was amplified from the embryonic library using EcoRI-tagged primers for exon 4 (5′-GCCGAATTCCATCCTGGAGCCGAGTGAA-3′) and exon 6 (5′-GACGAATTCTTCAGAGAGTCTAGCAGGGG-3′).
FIG. 1. Overview of the Evi12/Gnn locus on mouse chromosome 10. Evi12 is located upstream of Grp94 between the first exon and second intron of the novel Gnn gene. The locations of the 31 identified Gnn exons (short vertical lines above the map) are indicated, (more ...)
3′ RACE on cDNA from NFS107 cells using a primer set for exon 6 resulted in the identification of exons 7 and 8 as they are found in cDNA2. Primers for the first reaction were 5′-GCTCTTGTCTGGGGAACG-3′ and adapter primer, followed by 5′-GCTGTGGGAAGTCCACAC-3′ and adapter primer. The coding region of cDNA2 was amplified with primers for exon 4 and exon 8 using cDNA from NFS107 cells. For the primary PCR, the primers were 5′-CCTTGGCGACTGGGCCAGG-3′ and 5′-GCACCAGCAGGGGGCAGC-3′. For the nested PCR, the following primers were used: 5′-GCGACATCATCCTGGAGCC-3′ and 5′-ATCACACACCTGGAATCACGG-3′.
RT-PCRs on cDNA from NFS107 cells using primers for the coding region of Celera gene mCG8344, which is located downstream of Evi12 and cDNA1/2, resulted in identification of the 3′ end of cDNA3 (part of exon 16 to the end of exon 18). The primers were 5′-CAAGGAGAAGGCAGATACG-3′ and the adapter primer for the primary PCR and 5′-CCTCAGCGACACCTTGTGT-3′ and the adapter for the nested PCR. Subsequently, the complete cDNA3 coding sequence was amplified with primers for exon 4 and exon 18. The primary PCR was performed with 5′-CCTTGGCGACTGGGCCAGG-3′ and 5′-TTTATTCCTTATTGGGGTCATC-3′, and the nested PCR was performed with 5′-GCGACATCATCCTGGAGCC-3′ and 5′-TTTATTCCTTATTGGGGTCATC-3′. The Gnn/TU12B1-TY fusion cDNA4a was amplified from cDNA of NFS107 cells using primers for exon 6 and exon 19. The primary PCR was performed with 5′-GCTCTTGTCTGGGGAACG-3′ and 5′-GATGTCTGACAGGCTCATC-3′, and the nested PCR was performed with 5′-GCTGTGGGAAGTCCACAC-3′ and 5′-GTTCATGATGGATGGAACC-3′. Gnn/TU12B1-TY exons 19 to 31 (cDNA4b) were identified by RT-PCR using primers for exon 16 and in the last exon of the Celera mCG8347 gene. The primary PCR was performed with 5′-CAAGGAGAAGGCAGATACG-3′ and 5′-GGATTCTGGTCTGTTCGG-3′, and the nested PCR was performed with 5′-CCTCAGCGACACCTTGTG-3′ and 5′-CAAGCTCCCAAACTGGGC-3′.
To compare the expression of the Gnn/TU12B1-TY fusion and other Gnn products between NFS107 and control cell lines, we performed the RT-PCR that amplifies Gnn cDNA4a, and control PCRs that amplify the 3′ end of Gnn cDNA1 (exon 6) and the 3′ end (exons 16 to 18) of Gnn cDNA3 (see above).
All reactions with one exception were performed with Expand polymerase (Roche, Mannheim, Germany) under the following conditions: 30 cycles, with 1 cycle consisting of 1 min at 94°C, 1 min at 58°C, and 3 min at 72°C. The exception was amplification of the cDNA1 coding region, which was performed with Pfu polymerase (Promega, Madison, Wis.). The conditions for this reaction were as follows: (i) 3 cycles, with 1 cycle consisting of 1 min at 96°C, 1 min at 55°C, and 3 min at 72°C; (ii) 27 cycles, with 1 cycle consisting of 1 min at 96°C, 1 min at 60°C, and 3 min at 72°C.
Cloning of expression constructs.
The PCR product of cDNA1 was digested with EcoRI and either directly inserted into the EcoRI site of enhanced green fluorescent protein (EGFP)-C1 (BD Biosciences) to generate a construct encoding EGFP-Gnn1 or blunted and inserted in HpaI-digested PLNCX (BD Biosciences) to generate a nontagged construct encoding Gnn1. PCR products of cDNA2 and cDNA3 were first ligated into the TA cloning vector (Invitrogen, Breda, The Netherlands). The integrity of all three cloned PCR products was checked by sequencing. The correct clone of Gnn cDNA2 was excised with EcoRI and inserted into the EcoRI site of EGFP-C1 to generate a construct encoding EGFP-Gnn2. PLNCX-Gnn2 was generated by first blunting the EcoRI-excised Gnn cDNA2 and then inserting it in HpaI-digested PLNCX. All analyzed clones of Gnn cDNA3 in the TA cloning vector contained one or more single-nucleotide PCR errors, but each clone had a different error. One clone had an error in a region overlapping cDNA2. To generate the correct EGFP-cDNA3 fusion, TA-cDNA2 was digested with EcoRI and SstI, and TA-cDNA3 was partially digested with SstI and EcoRV. The resulting fragments were ligated simultaneously into EcoRI/SmaI-digested EGFP-C1 to generate EGFP-Gnn3. To generate PLNCX-Gnn3, the two fragments were simultaneously ligated into HpaI-digested PLNCX.
PCR products cloned in the TA vector were sequenced using an ABI 3100 sequencer (Perkin Elmer, Nieuwerkerk a/d IJssel, The Netherlands) with forward and reverse primers (from the TA cloning kit) and Gnn-specific primers.
Transfection and immunofluorescence.
HEK-293 cells were grown on glass coverslips and transfected by the calcium phosphate transfection method. Two days after transfection, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4°C for 20 min. Fixed cells were washed three times with PBS, embedded in Vectashield (Vector Laboratories, Burlingame, Calif.), and observed by confocal laser scanning microscopy.
Generation of Gnn antibody.
An affinity-purified rabbit antiserum against Gnn was prepared by Research Genetics (Huntsville, Ala.). The antiserum was raised against the N-terminal DFQEERDFLAKSIFPNLD sequence of Gnn, which is 100% conserved in mice and humans.
Cell pellets or homogenized tissues were lysed in ice-cold lysis buffer (50 mM Tris-HCl [pH 8], 100 mM NaCl, 1% Triton X-100, 1% Pefabloc SC, 50 μg of aprotinin/ml). Protein samples were run on 5 to 8% acryl amide gels and blotted on nitrocellulose. Blots were blocked overnight in 5% milk and incubated overnight with anti-Gnn antibody (diluted 1:1,000). Proteins were detected with horseradish peroxidase-conjugated swine anti-rabbit serum (DAKO, Glostrup, Denmark), followed by an enhanced luminescence reaction.
Cytospins were fixed with acetone, treated for 10 min with 0.5% hydrogen peroxide in PBS to block endogenous peroxidases, washed in PBS containing 0.05% Tween 20 (PBT), and blocked for 30 min in PBT containing 1% bovine serum albumin. Samples were incubated overnight with anti-Gnn antibody (diluted 1:100) in blocking buffer. Horseradish peroxidase-conjugated swine anti-rabbit antibody was used as the secondary antibody. Samples were developed with diaminobenzidine and enclosed in Entellan (Electron Microscopy Sciences, Hatfield, Pa.).
Quantitative RT-PCR on randomly primed cDNA was performed as previously described (22
). The primers used for amplification of Gnn
sequences were 5′-CAAAGGGCTTCCTGTCAGATT-3′ and 5′-TGCTTGCAGTTCTCCACAAA-3′ for the product of exons 4 and 5 and 5′-CTGAATCCAGACGCCATTTT-3′ and 5′-ATGGCAAAGCTTGGGTCATA-3′ for the product of exons 19 and 20.
Web links for sequence analysis. Nucleotide sequence accession numbers.
Gnn sequences have been deposited in GenBank with the following accession numbers: AY651019 (Gnn cDNA1), AY651020 (Gnn cDNA2), AY651021 (Gnn cDNA3), and AY651022 (Gnn cDNA4b).