SUPPLEMENTAL MATERIAL

Transcription

1 SUPPLEMENTAL MATERIAL MATERIALS AND METHODS Generation of TSPOΔ/Δ murine embryonic fibroblasts Embryos were harvested from 13.5-day pregnant TSPOfl/fl mice. After dissection to eviscerate and remove the head, embryos were minced and pieces were allowed to attach and grow in a 5% CO 2 incubator to reach confluence. After the first passage, cells were infected with approximately 10 MOI of a cre recombinase expressing Adenovirus (Vector Biolabs), to induce recombination and generate TSPOΔ/Δ fibroblasts. Cells were passaged for a second time, allowed to grow for 48 hours and then used for DNA extraction. PCR amplification of the region spanning the recombination site from intron 1 to exon 4 was performed using specific primers (Supplemental Table 5), in both TSPOfl/fl and TSPOΔ/Δ fibroblasts (Supplemental Figure 1). TOPO TA cloning and sequencing PCR amplified product (872 bp) from genomic DNA extracted from TSPOΔ/Δ fibroblasts was cloned into a TOPO TA vector (pcr2.1-topo, Life Technologies). Universal sequencing primers included in the plasmid backbone was used to sequence the inserted product (Biotechnology Resource Center, Cornell University). Sequenced PCR product was aligned to the predicted design of the recombined TSPO vector and verified for deletion of Exons 2 and 3 (Supplemental Figure 1).

2 SUPPLEMENTAL TABLES Supplemental Table 1. TSPO allele targeted ES cell clone validation long-range PCR primers Primer/Probe Sequence Long-range PCR LR-TSPO-F1 5 GGGTCTCATGCTTGCTCTGATCAT 3 LR-TSPO-vR 5 CCGTCGTTTTACAACGTCGTGACT 3 Loss of allele at outside loxp TSPO-Fp 5 CAGAGGCAGAAGGATTGCTATAAGTC 3 TSPO-Rp 5 TGTGTAGCCTTAGCTGATTTTGAACT 3 TSPO probe FAM 5 CAGCCTGGTCTATAAAG 3 MGB Neo copy number Neo-Fp 5 CCATTCGACCACCAAGCG 3 Neo-Rp 5 AAGACCGGCTTCCATCCG 3 Neo probe FAM 5 AACATCGCATCGAGCGAGCACGT 3 TAMRA

3 Supplemental Table 2. Genotyping primers Primer Sequence TSPO genotyping P1 5 GTACCAGCAGAGGCAGAAGG 3 P2 5 ATGCCCACCTCTGTTCTCAG 3 P3 5 CGCCATCTCAGCACTTTACA 3 Cre genotyping Cre3 5 CGTTCACCGGCATCAACGTTT 3 Cre5 5 GCGGCATGGTGCAAGTTGAAT 3 Int-ControlF 5 CTAGGCCACAGAATTGAAAGATCT 3 Int-ControlR 5 GTAGGTGGAAATTCTAGCATCATCC 3

4 Supplemental Table 3. RT-PCR Primers for TSPO cdna Primer Region Sequence E1F Exon 1 5 CAGTGTCCTTCACGGAACAA 3 E2R Exon 2 5 TGAATACAGTGTGCCCCAGA 3 E2F Exon 2 5 ATCAGTTGCAATCACCATGC 3 E3R Exon 3 5 TGTGAAACCTCCCAGCTCTT 3 E3F Exon 3 5 TGGGAGGTTTCACAGAGGAC 3 E4R Exon 4 5 AACCTACCTGGTGGCTTCCT 3

5 Supplemental Table 4. Plasma hormone levels in TSPOfl/fl and TSPOcΔ/Δ female mice Hormone Mean plasma hormone concentrations ± SE* Non-pregnant 14.5-day Pregnant TSPOfl/fl TSPOcΔ/Δ TSPOfl/fl TSPOcΔ/Δ Estradiol (pg/ml) ± ± ± ± 2.42 Progesterone (ng/ml) 4.83 ± ± ± ± 4.47 * No significant differences were detected between TSPOfl/fl and TSPO-Amhr2cre/+mice.

6 Supplemental Table 5. Primers for PCR and sequencing the TSPO locus Primer Region Sequence Int1F Intron 1 5 TCACCAAGGGTGTGAATGAA 3 E4R Exon 4 5 AACCTACCTGGTGGCTTCCT 3 M13F Plasmid 5 GTAAAACGACGGCCAG 3 M13R Plasmid 5 CAGGAAACAGCTATGAC 3

7 SUPPLEMENTAL FIGURES Supplemental Figure 1 A TSPO Recombination - exons and primers Exons: fl B TSPO fl/fl -/ Int-1F Cre recombinase - Exon 2,3 deleted Int-1F P3 P2 P3 P2 C Sequence alignment of 872 bp product with TSPO / locus E4R E4R r TSPO locus PCR product FRT site LoxP site Exon 4 Supplemental Figure 1. Deletion of TSPO exons 2 and 3 in the recombined genomic locus. (A) Schematic showing cre-mediated recombination at the TSPOfl/fl locus. Location of primers used for amplification and sequencing are indicated (for sequences, see Supplemental Tables 2 and 5). (B) PCR amplification showing recombination in the TSPOΔ/Δ locus. Primers spanning from Intron 1 (Int1F) to Exon 4 (E4R) show that TSPOfl/fl genotype results in an expected 2697 bp band that is reduced to an 872 bp band in the TSPOΔ/Δ genotype. Internal control primers P2-P3 within intron 3 show a 161 bp band in both genotypes. (C) Sequence for the 872 bp product obtained in the TSPOΔ/Δ genotype aligns perfectly with the predicted TSPO locus after deletion of exons 2 and 3. PCR product sequence indicates the presence of the FRT site, one LoxP site and Exon 4, confirming the deletion of exons 2 and 3 in the TSPOΔ/Δ genotype.

8 Supplemental Figure 2 Supplemental Figure 2. Amhr2cre/+-mediated gene deletion in the ovary. (A) ROSA26-tdTomato (R26tdTom) reporter mouse ovary showing recombination in the corpus luteum and interstitial cells in R26tdTom-Amhr2cre/+ mice. Scale bar 50 µm. (B) Genotyping for Amhr2cre/+ mice using specific primers [Con-internal control].

9 Supplemental Figure 3 Supplemental Figure 3. TSPO deletion in TSPOcΔ/Δ mouse ovary. (A) Immunohistochemical localization showing a decrease in TSPO positive cells in TSPOcΔ/Δ ovaries. Hematoxylin and eosin staining showing unaltered ovarian functional morphology in TSPOcΔ/Δ mice. Scale bar 50 µm. (B) Western blots showed a decrease in TSPO protein in TSPOcΔ/Δ ovarian tissue (n=3).

10 Supplemental Figure 4 Supplemental Figure 4. TSPO deletion does not affect expression of genes involved in ovarian steroidogenesis. (A) TSPO expression was decreased in TSPOcΔ/Δ ovaries. (B) StAR, (C) CYP11A1, and (D) HSDB1 expression levels were similar between TSPOfl/fl and TSPOcΔ/Δ ovaries. (E) TSPO2 expression was not detectable in TSPOfl/fl and TSPOcΔ/Δ ovary. Femur bone marrow was used as a positive control. (Mean ± SEM; ND*: not detected; n=6/group)