Capel Group Protocols
- 1. Embryos were removed at 11.5, 12.5, and 13.5 dpc from wild type CD-1 females that were time-mated to the following reporter lines, with the following exceptions:
- a. Sry-EGFP: labels supporting cells in the male at 11.5 dpc, and in the female at all stages (Albrecht and Eicher, 2001).
- b. Sox9-ECFP: labels male supporting cells at 12.5 and 13.5 dpc (Kim et al., 2007).
- c. αSma-EYFP: labels interstitial cells. CD-1 females were not used, and instead homozygous αSma-EYFP FVB females were used to increase the intensity of the fluorescent label (Cool et al., 2008).
- d. Mafb-EGFP: labels interstitial cells. This is a knock in of GFP into the Mafb locus. A heterozygous male was mated to a wild type CD-1, and only embryos with GFP-positive gonads were used for sorting. Thus, all the embryos used were heterozygous for Mafb (Moriguchi et al., 2006).
- e. Flk1-mcherry: labels endothelial cells. CD-1 females homozygous for the Flk1-mcherry transgene were used to increase the intensity of the fluorescent label (Larina et al., 2009).
- f. Oct4-EGFP: labels germ cells (Szabo et al., 2002).
- 2. For 12.5-13.5 dpc embryos, the sex of the gonad is obvious, thus no genotyping was required, and we proceeded with the dissection. For 11.5 dpc embryos, where sex is not apparent, the tails were removed for genotyping to determine sex while the embryos were stored at 4ºC in a 24 well plate in PBS.
- a. Each tail was placed in 200 µl of 50 mM NaOH and heated to 95°C for 10 minutes, then vortexed briefly.
- b. 50 µl of 1M Tris HCl pH 7.6 was added and the samples were vortexed briefly.
- c. PCR was performed with an annealing temperature of 55ºC to detect Kdm5c (X chromosome) and Kdm5d (Y chromosome) with the primers 5′-TGAAGCTTTTGGCTTTGAG-3′ and 5′-CCGCTGCCAAATTCTTTGG-3′. Females have a single band of 320 bp, and males have two bands of 320 bp and 280 bp. These bands are resolved on a 2.5% agarose gel.
- 3. The urogential ridge and dorsal aorta were removed from the embryo, and then the gonad/mesonephric complex was removed. The gonad was then removed from the mesonephros, with the following exceptions:
- a. For Oct4-EGFP sorts, the mesonephros was left attached. Oct4 expression is highly specific to the germ cells, and so removing the mesonephros provided no benefit.
- b. For 11.5 dpc Flk1-mcherry sorts, only the anterior and posterior portions of the mesonephros were removed by cutting at a 45° angle from the end of the gonad. The vasculature in the gonad arises from a plexus in the mesonephros. Because we believe this is one population of endothelial cells at 11.5 dpc, the plexus was included.
- 4. All the male gonads were pooled into one 1.7mL tube, and the female gonads were pooled into a separate 1.7 mL tube.
- 5. The gonads were incubated in 250 µl 0.25% Trypsin EDTA (Gibco #25200) at 37°C for 5-10 minutes.
- 6. As much Trypsin as possible was removed without disturbing the tissue, which naturally settles to the bottom of the tube.
- 7. We added 400 µl PBS (without Ca/Mg) with 4 µl RNase-free DNase (Promega #M6101) to each tube. However, DNase was not used for the supporting cells (Sry-EGFP and Sox9-ECFP) because this reduced the yield.
- 8. The cells were dissociated by pipetting up and down with a P200 set at 100 µl, and then pulling the cells through a 27 gauge needle until no clumps were visible (around 5x).
- 9. The cells were passed through a cell strainer (BD Falcon #352235) by tapping, and then centrifuging for 1 second at 1000 rpm to collect the remaining liquid.
- 10. The cells were taken to the Duke Comprehensive Cancer Center Flow Cytometry Shared Resource for FACS sorting.
- 11. The positive fraction from the cell sort was pelleted at 6000 rpm for 5 minutes, and the liquid was removed.
- 12. The cells were immediately frozen at -80°C.
RNA extraction and sample preparation for Affymetrix Gene 1.0 ST arrays
- 1. For many of the cell types, multiple sorts were pooled to collect enough cells. RNA was extracted from over 100,000 cells to as few as 10,000 cells.
- 2. RNA was prepared using the RNeasy Micro kit (Qiagen #74004) following manufacturer instructions for “Cells,” with the following exceptions:
- a. The protocol was started at step 2 (disruption with RLT). β-ME was not added. To disrupt the cells, they were pipetted with a P1000, vortexed for 5 minutes, and centrifuged for 2 seconds. When multiple tubes of cells were pooled, the first was disrupted, the RLT from the first tube was moved to the second one, the cells were disrupted in the second tube following the same method, and this was repeated until all the cells were pooled.
- b. Step 3 (homogenization) was skipped.
- c. In step 10, samples were washed 3x with RPE, rather than one.
- 3. Samples were prepared for the Affymetrix Gene 1.0 ST arrays using the Nugen WT-Ovation Pico RNA Amplification System (3300), WT-Ovation Exon Module (2000), and the Encore Biotin Module (4200), following manufacturer instructions. For purification following the Pico and Exon kits, the Qiagen QIAquick PCR Purification Kit (28104) was used following the instructions provided by Nugen.
- 4. Fragmented and labeled product was submitted to the Duke Institute for Genome Sciences and Policy Microarray Facility for hybridization.
Albrecht, K.H., and Eicher, E.M. (2001). Evidence that Sry is expressed in pre-Sertoli cells and Sertoli and granulosa cells have a common precursor. Dev Biol 240, 92-107.
Cool, J., Carmona, F.D., Szucsik, J.C., and Capel, B. (2008). Peritubular myoid cells are not the migrating population required for testis cord formation in the XY gonad. Sex Dev 2, 128-133.
Kim, Y., Bingham, N., Sekido, R., Parker, K.L., Lovell-Badge, R., and Capel, B. (2007). Fibroblast growth factor receptor 2 regulates proliferation and Sertoli differentiation during male sex determination. Proc Natl Acad Sci U S A 104, 16558-16563.
Larina, I.V., Shen, W., Kelly, O.G., Hadjantonakis, A.K., Baron, M.H., and Dickinson, M.E. (2009). A membrane associated mCherry fluorescent reporter line for studying vascular remodeling and cardiac function during murine embryonic development. Anat Rec (Hoboken) 292, 333-341.
Moriguchi, T., Hamada, M., Morito, N., Terunuma, T., Hasegawa, K., Zhang, C., Yokomizo, T., Esaki, R., Kuroda, E., Yoh, K., et al. (2006). MafB is essential for renal development and F4/80 expression in macrophages. Mol Cell Biol 26, 5715-5727.
Szabo, P.E., Hubner, K., Scholer, H., and Mann, J.R. (2002). Allele-specific expression of imprinted genes in mouse migratory primordial germ cells. Mech Dev 115, 157-160.