FIGURE 2. Cdx2-deficient blastocysts and ES cell derivation.

a, Cdx2 immunostaining of day 3.5–4.5 wild-type and nuclear transfer blastocysts. The following donor cells were used for the nuclear transfer (from second column, left to right): Cdx2^2Lox tail-tip, Cdx2^2Lox ES cells, and Cdx2^1Lox ES cells. b, A typical Cdx2^2Lox tail-tip nuclear transfer blastocyst is shown 84 h after activation of the reconstructed oocytes. Cdx2-deficient blastocysts initially cavitated but failed to maintain the blastocoel and collapsed. Below, an expanded nuclear transfer blastocyst derived from control cells is shown. c, RT–PCR analysis of normal and Cdx2-deficient nuclear transfer pre-implantation morula/blastocysts. Four 4-cell embryos were pooled and RNA was extracted for reverse transcription. All other samples were prepared from single morulae or blastocysts. Tail-tip fibroblasts (lane 6) express neither Cdx2 nor Oct-4. Trophectoderm stem (TS) cells (lane 7) express Cdx2, but no Oct-4. A faint Cdx2-specific band, such as that seen in the blastocyst containing the shRNA construct targeting Cdx2 shown in the figure, was detected in less than half of the tested embryos; most gave no signal in this test. d, Derivation of ES cells from Cdx2-deficient blastocysts. A Cdx2^2Lox tail-tip nuclear transfer-derived blastocyst with its initial outgrowth after 72 h (left) and a wild-type blastocyst (right) with its initial outgrowth are shown.