Plko 3g

shRNA sequences for NgR1 and NgR2 were obtained of Sigma-Aldrich (St. Louis, MO, USA, Mission shRNA). These sequences were ligated to the eGFP-expressing lentiviral vector pLKO.3G (Addgene, Cambridge, MA, USA) via sites EcoRI and PacI. We used as control shRNA ‘scrambled' sequences designed using the siRNA Wizard Program (InvivoGen, San Diego, CA, USA). Oligonucleotide shRNA sequences were as follows: NgR1: 5′-AATTCTCTACCTACAAGACAACAAT-3′, NgR2:5′-AATTGGTCAGCCTACAGTACCTCTA-3′ and scrambled: 5′-CCTAAGGTTAAGTCGCCCTCGCTCG-3′. Lentiviral vectors were produced by transient cotransfection of human embryonic kidney 293T cells in DMEM containing 10% FBS, using the recombinant plasmid-pLKO.3G carrying transgene sequences, sequences encoding helper (packaging) PsPax2 (Addgene) functions and sequences encoding Env glycoproteins PMD2.G (Addgene). At 48–60 h postransfection, lentiviral vector stocks were concentrated by ultracentrifugation and titrated by flow cytometric (FACS) methods via infection of HEK 293 T cells.
Differentiated MN1 cells were infected by addition of lentiviral particles carrying shRNA sequences for NgR1 and NgR2 at 1 × 10⁴ pfu for 4 days. MN1 cells infected with the virus were identified by eGFP expression. Expression levels of the receptors were monitored by western blotting with receptor-specific antibodies.

Gata-DKO and control TSCs were transfected with pLKO.3G (Addgene plasmid 14748) and cell sorted (flow cytometry is described in the supplementary Materials and Methods) for strong GFP expression. Sorted cells were cultured in the presence or absence of tamoxifen. Gene deletions were confirmed by PCR. Eight to ten TSCs were microinjected into morulae or very early stage blastocysts using standard techniques. Embryos were allowed to grow to expanded blastocyst stage and micrographed for GFP fluorescence.