C57bl 6 female

Female C57BL/6 mice served as recipients of ID8 tumors. They were obtained commercially (Jackson Laboratory, Bar Harbor, ME) at six weeks of age and immunized at 7–10 weeks of age by subcutaneous injection in the abdominal flanks with 100 μg of recombinant mouse AMHR2-CD in 200 μL of an emulsion of equal volumes of water and complete Freund's adjuvant (CFA, Difco, Detroit, MI) containing 400 μg of Mycobacteria tuberculosis. TgMlSIIR-TAg (DR26 line) transgenic mice (provided by DDC) were maintained by breeding male TgMISIIR-TAg (H-2^b) mice to wild-type syngeneic C57BL/6 females (Jackson Laboratory). TgMlSIIR-TAg mice were immunized at 6-7 weeks of age with 100 μg of recombinant mouse AMHR2-CD in CFA as described above. To determine fertility phenotypes, age-matched test and control vaccinated C57BL/6 female mice were mated with the same C57BL/6 males. All protocols were preapproved by Cleveland Clinic's Institutional Animal Care and Use Committee.

MSCs were derived from C57BL/6 wild type, Pml^−/− and Pml^F/FPrrx1-Cre. To generate Pml^F/F mice, the Pml genomic sequence was cloned by PCR, and then inserted into the pEZ-LOX-FRT-DT vector. The construct was linearized with BamHI and electroporated into embryonic stem cells. Transfectants were selected in G418 (350 μg/ml) and ganciclovir (2 μM) and expanded for Southern blot analysis. Chimeric mice were produced and then mated with C57BL/6 females (The Jackson Laboratories) (for more details see  Chen et al.³⁴ ). Prrx1-Cre mice were purchased from the Jackson Laboratory. For all the in vivo experiments, mice with matched sex and age were used, and were randomly assigned to experimental groups. No animal was excluded from the final experimental analysis; the investigators were not blinded to group allocation. Animal experiments were performed in accordance with the guidelines of Beth Israel Deaconess Medical Center Institutional Animal Care and Use Committee.

KH2 ESCs were cultured in standard serum/LIF conditions as described previously (Meissner et al., 2009 (link)). Targeting of the Dnmt3b cDNAs into the Col1A1 locus of KH2 cells was conducted using the gene targeting kit from Open Biosystems based on the original publication (Beard et al., 2006 (link)). Briefly Dnmt3b1 cDNAs were cloned into the pBS31’-RBGpA vector and electroporated (2 pulses at 500 V and 25 μF on Bio-Rad Gene Pulser II) into the KH2 cells along with pCAGGS FLPe recombinase plasmid. Clones with successful integration of the targeting vector were identified though hygromycin (140 μg/ml) selection for 10 days.
To assess induction in the targeted ESCs, 1 mM retinoic acid (Sigma) was added for 6 days to induce differentiation and as a consequence decrease the endogenous Dnmt3b expression level.
The Dnmt3b targeted ESCs were used to generate mice through diploid blastocyst injections (HSCI Genome Modification Facility). Chimeras were backcrossed to C57BL/6 females (The Jackson Laboratory) to obtain F1 heterozygous transgenic mice. For genotyping, DNA was isolated from tail biopsies and subjected to PCR using primers and conditions as previously described (Beard et al., 2006 (link)).
For transgene induction, mice were fed 1 mg/mL doxycycline in the drinking water supplemented with 10 mg/mL sucrose for the specified duration.