Supplemental Data
Files in this Data Supplement:
- Supplemental Figure S5 - "Adult GT3/GT3 tubules are devoid of KIT+ spermatogonia. Sections of paraffin-embedded testes from adult (12-wk-old) mice were subjected to immunohistochemical analysis using an antibody specific for KIT. A and C) In +/+ testis KIT staining was observed at the plasma membrane of spermatogonia, as expected. Insets show magnified images of demarcated regions. B and D) GT3/GT3 tubules contain some KIT+ Leydig cells (*), but no KIT+ spermatogonia. All images were taken under 10 × magnification."
- Supplemental Figure S3 - "Assessment of the frequency with which the GT3 gene trap is spliced around, generating full-length Vrk1 mRNA, in 4-wk-old mice. Tissues were harvested from 4-wk-old +/+ and GT3/GT3 mice. Vrk1-specific cDNA sequences (as well as a &[beta]-actin control) were amplified by semi-quantitative RT-PCR using the primers shown in the Figure 3C schematic. The relative level of total Vrk1 mRNA in +/+ versus GT3/GT3 tissues was calculated (upper panels); the fold difference is shown beneath the appropriate lanes. The fold-difference in the level of the full-length Vrk1 mRNA, generated by splicing around the gene-trap, in +/+ versus GT3/GT3 tissues (shown beneath middle panels) was calculated after normalizing to the total Vrk1 mRNA levels found in each tissue."
- Supplemental Figure S6 - "Expression of Kit ligand is relatively unchanged in GT3/GT3 mice. A) RNA was harvested from 4-, 7-, and 16-wk old +/+ and GT3/GT3 tissues. Vrk1 and Kit ligand (Kitl) cDNA sequences were amplified by semi-quantitative RT-PCR. Vrk1-specific PCR was performed as a positive control, using primers which anneal downstream of the gene trap to demonstrate that GT3/GT3 tissues displayed the loss of full length Vrk1 characteristic to GT3/GT3 mice. B) The relative level of total Kitl mRNA in GT3/GT3 versus +/+ tissues was calculated; the fold difference is shown graphically."
- Supplemental Figure S4 - "VRK1 protein is most highly expressed in proliferative tissues and is lost in GT3/GT3 mice. Immunoblot analysis was performed on liver, spleen and testis harvested from 4-wk and 14-wk-old mice; whole cell lysates were prepared and 100 µg of protein was subjected to electrophoretic fractionation. Immunoblot analysis was performed using a polyclonal antibody raised against an N-terminal VRK1 peptide (amino acids 3-18). Multiple species of VRK1 were seen in +/+ samples (open arrowheads) and are missing from GT3/GT3 samples; a cross-reacting protein that is not affected by the GT3 insertion is indicated by the filled arrowhead. Identical spleen and testis lysates were analyzed in parallel with a commercially available VRK1 antibody (shown below lanes 9-12), demonstrating that similar banding patterns are observed with a second antibody."
- Supplemental Figure S1 - "Genotypic analysis of the murine GT12 Vrk1 locus. A and B). ScaI-digested genomic DNA was subjected to Southern blot analysis (B) with cDNA probes spanning exons 11-15 (grey font in A). The ScaI restriction sites are indicated by grey triangles; black and gray horizontal lines highlight the predicted restriction fragments. The sizes of these fragments are shown above the lines. B) Southern blot analysis. Representative Southern blots of genomic DNA show the wild type (+/+), heterozygous (+/GT) and homozygous (GT/GT) Vrk1 locus in animals containing the GT12 gene-trap insertions. Blots were probed with cDNA fragments described above. The arrows at the right of each panel indicate the wild-type and expected targeted fragments. C and D) The differences between the expected and actual results from the Southern analysis are best explained by a tandem tail-to-tail insertion of the βgeo trap into the Vrk1 locus of GT12/GT12 mice. Schematic representation of the wild type Vrk1 locus and the GT12 locus if it contained the tail-to-tail insertion of the βgeo cassette is shown (C). ScaI and KpnI restriction sites (grey and black arrowheads, respectively) and the corresponding fragment sizes resulting from these digests are shown. Genomic DNA from tail tips of +/+ and GT12/GT12 animals was digested with KpnI or ScaI and subjected to Southern blot analysis (D). Membranes were probed with a probe representing intron 12 of the Vrk1 locus (P1) or a βgeo probe (P2; shown in C). The fragment sizes expected for the +/+ sample, or for the GT12/GT12 sample if a tail-to-tail insertion of the gene trap had occurred, are shown below the blot. N/A applies to the +/+ samples in which no fragment should be detected by the &[beta]geo specific probe."
- Supplemental Figure S2 - "Genotypic analysis of the murine GT12 Vrk1 locus. ScaI-digested genomic DNA was subjected to Southern blot analysis (B) with cDNA probes spanning exons 2-6 (grey font in A). The ScaI restriction sites are indicated by grey triangles; black and gray horizontal lines highlight the predicted restriction fragments. The sizes of these fragments are shown above the lines. B) Southern blot analysis. Representative Southern blots of genomic DNA show the wild type (+/+), heterozygous (+/GT) and homozygous (GT/GT) Vrk1 locus in animals containing the GT12 gene-trap insertions. Blots were probed with cDNA fragments described in the legend for A. The arrows at the right of each panel indicate the wild-type and expected targeted fragments. An additional band is seen that is likely to correspond to a 2.9 kb ScaI fragment found on chromosome 19; this fragment has 87% identity to the Upstream Probe. The absence of an in frame ATG and the presence of multiple stop codons within this fragment suggests that it may be a degenerate Vrk1 pseudogene."