FIGURE 1. Derivation of NT-ESCs from Cdx2-deficient blastocysts.

a, Primary tail-tip fibroblasts were infected with a conditional lentiviral RNA interference (RNAi) construct targeting Cdx2 before nuclear transfer (NT). Blastocysts deficient for Cdx2 were morphologically abnormal and unable to implant but gave rise to NT-ESCs. After initial expansion of the Cdx2 knockdown NT-ESCs (Cdx2^2Lox) we used transient Cre expression to generate subclones (Cdx2^1Lox) with a deleted hairpin. To test the potency of ES lines before and after 'loop-out' we used teratoma formation, diploid and tetraploid blastocyst injections as well as nuclear transfer. b, The conditional RNAi system (pSicoR) has been described previously⁶. The shRNA, which targets nucleotides 1890–1908 located in the 3' UTR of Cdx2, was cloned into the conditional RNAi vector generating pSicoR-Cdx2^2Lox. This vector carries the Cdx2 shRNA construct and an enhanced green fluorescence protein (EGFP) gene flanked by two LoxP sites (2Lox), which allows for Cre-mediated deletion of the shRNA and the EGFP sequences.