Skip to main content
. Author manuscript; available in PMC: 2014 Apr 25.
Published in final edited form as: Nat Commun. 2011 Jan 11;2:153. doi: 10.1038/ncomms1153

Figure 2. Molecular cloning of two alternative splice variants of CatSperδ.

Figure 2

(a) Tissue distribution of CatSperδ mRNA by reverse-transcription PCR. CatSperδ (upper) and Glyceraldehyde-3-Phosphate Dehydrogenase (control; lower) from 12 mouse cDNAs; negative control (lane “−”). CatSperδ was detected only in testis. (b and c) Molecular cloning of CatSperδ cDNAs. Two bands differing by 118 bp were amplified by mouse testis first-strand cDNAs by PCR with primers corresponding to the most upstream 5′ sequence identified by 5′-RACE and 2 different gene-specific primers (GSP) nested at the 5′-UTR of Tmem146-s. RT, reverse transcriptase (b). Whole open reading frames (ORFs) of Tmem146 were amplified from testis cDNA (c). (d) Schematic diagram of CatSperδ splice variants. Two alternatively spliced mRNA variants are transcribed from the Tmem146 gene. Tmem146-s has a start site in exon 7 (blue). Tmem146-l contains a new start site in exon 1 (green) due to a change in the ORF by the additional 118 bp exon 5 (orange). The locations of probes for in situ hybridization are illustrated above the transcripts. Probe 1 is complementary to both Tmem146-s and –l. Probe 2 was amplified from Tmem146-s cDNA and corresponds to the splicing region (spanning exon 4 and 6). (e) Heterologous expression of CATSPERδ isoforms. V5-tagged Tmem146-s or Tmem146-l, cDNAs were transfected into HEK293T cells. After immunoprecipitation with anti-V5, immune complexes were probed with anti-V5. (f) Spatial localization of Tmem146 splice variants. Representative fields of in situ hybridization in mouse testis using antisense 1 (upper left) and antisense 2 (lower left). Sense probes served as background controls (right panels). Scale bar, 100 μm. (g) Temporal Tmem146-s and Tmem146-l mRNA levels (real time RT-PCR) during testis postnatal development. mRNAs are normalized to TATA binding protein (TBP) at each time point and expressed as ratios relative to adult (80-day) mouse testis (± SEM compared to each prior time point *P < 0.05, **P < 0.005). Tmem146-s mRNA was detected at 17 days (unpaired two-tailed t-test, P = 0.0017) while Tmem146-l appeared at 20 days (P = 0.047 at 20 days, P = 0.042 at 23 days), consistent with the in situ hybridization experiments. Results are from 3 independent experiments.