Generation and Characterization of 2C::tomato ES Cells

2C∷tomato was created by digesting the MERVL LTR-Gag clone #9²⁹ with MluI and HindIII, resulting in MERVL LTR 1-730, and was ligated into pcDNA3 hygro tdtomato with CMV promoter removed. To generate 2C∷tomato ES cells, Kdm1a Fl/Fl; Cre:ERT ES cells were transfected with 2C∷tomato using Lipofectamine 2000 (Invitrogen) and selected with 150ug/ml hygromycin for 7 days. Colonies containing tomato⁺ cells were then picked and expanded. 2C∷ERT2-Cre-ERT2 was generated by replacing tdtomato with an ERT2-Cre-ERT2 insert using EcoR1 and Not1 sites. DNA was linearized with Mlu1 and AvrII sites before injection into embryos to generate transgenic mice. The resulting mice were mated with ROSA∷LSL-tomato mice (JAX 007905), ROSA∷LSL-DTA mice (JAX 010527), or ROSA∷LSL-LacZ mice (Gift of Anderson lab), and ES lines were derived using standard procedures. Kdm1a GT/GT, Kdm1a Fl/Fl, KAP1 ES3 Cre, and G9A TT2 ES cells were described previously^{29 –31 (link)}. RNA-Seq from oocytes and 2C embryos was performed by lysing litters of embryos (5–10 embryos) in Prelude Direct lysis buffer (Nugen) and amplifying RNA using the Ovation RNA-Seq system (Nugen) before library construction using the Tru-Seq RNA sample prep kit (Illumina). Microarray, QRT-PCR, immunostaining, and chimeric mouse injections were performed as described²⁹ . All animal experiments we performed in accordance with the Salk Institute's IACUC guidelines.