Supplementary Information for
Hu YC, de Rooij DG, Page DC.
Tumor suppressor gene Rb is required for self-renewal of spermatogonial stem cells in mice.
PNAS 110, 12685 (2013)
- Figure S1 Generation ofmouse vasa homolog(MvhCre-mOrange/+)mice.(A) Schematic representation of knock-in ofCre-mOrangegene into theMvhlocus. The targetedknock-in allele was generated by homologous recombination of the targeting vector. The FLPe recombinase recognition target (FRT)-flankedNeocassette was excisedby further mating toACTB-FLPemice (Jackson Laboratory, stock number 005703).Neo, neomycin gene; pA, polyadenylation signal;PGK, phosphoglycerate kinasepromoter; SA, splicing acceptor. (B) Southern analysis of 5'junction of the knock-in (ki) allele. Genomic DNA from targeted ES cells was digested withBgl IIenzyme andhybridized with a P32-labeled 5'probe external to the targeted area. (C) PCR analysis of 3'junction of the targeted allele in ES cells, using the primers as indicated. (D)Successful excision of the FRT-flankedNeocassetteinmicewasconfirmed by PCR analysis, using the primers as indicated. (E) Primer sequence information. (F) The Crerecombination activity became apparent in the gonads of embryonic day (E) 15.5 embryos that carriedMvhCre-mOrange/+and a ROSA26-lacZ reporter. The Cre activitywas assessed by the presence ofß-galactosidase expression using X-Gal staining (dark blue, arrows). (Right) Higher magnification of boxed area in left panel.
- Figure S2 Unmarked periodic acid-Schiffstained images for Fig. 3B. (Scale bar, 25ĩm.) P, postnatal day.
- Figure S3 Number of preleptotene spermatocytes per 1,000 Sertoli cells in stage VII tubule cross-sections of control and retinoblastoma geneRbconditionalknockout (RbcKO,Rbfl/fl;MvhCre-mOrange/+) testes (meanąSD,n=3 for all points, exceptn=2 for P40 controls). **P<0.01 (two-tailed Studentttest).
- Figure S4 Increased expansion of spermatogonia inRbcKO testes was observed at P28 and P40, when spermatogonia were counted only in those stage VIItubule cross-sections that retained spermatogonia (meanąSD,n=4 for P28,n=2 for P40). *P<0.05 (two-tailed Wilcoxon test). Note that there were initiallyfour P40RbcKO mice whose testes were examined; two of these four mice were excluded from the count because their testes displayed no spermatogonia inany stage VII tubule cross-sections examined.
- Figure S5 Higher cell death index inRbcKO testes than controls at P10. The apoptotic cells were visualized by staining for active caspase-3. The level ofapoptotic cells was counted only in the tubule cross-section that contained apoptotic cells in control andRbcKO testes (meanąSD,n=4 for each group).*P<0.05 (two-tailed Student t test)