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First published online 18 April 2006
doi: 10.1242/jcs.02913

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Inefficient reprogramming of the hematopoietic stem cell genome following nuclear transfer

¹ RIKEN Bioresource Center, Tsukuba, Ibaraki 305-0074, Japan
² Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba 277-8562, Japan

View larger version (85K): [in a new window]   Fig. 1. Two pups cloned from (B6x129)F1 HSCs. The pups were normal in appearance and showed active movement shortly after caesarian section, but were cannibalized by the foster mothers within 24 hours. They had enlarged placentas, as observed for other somatically cloned mouse pups (Inoue et al., 2002).

View larger version (34K): [in a new window]   Fig. 2. Transcriptional activity of 2-cell HSC clone embryos assessed by a BrUTP incorporation assay. (A) Incorporation of BrUTP was detected by a specific antibody in the nuclei (arrows) of cumulus clone embryos (left), HSC clone embryos (right) and parthenogenetic embryos (not shown) at 12 hours post-activation. (B) The fluorescent intensity in the nuclei, which depicts transcriptional activity, was not different between cumulus and HSC clone embryos (P>0.05), and both were at the control level (parthenogenetic embryos=100%).

View larger version (19K): [in a new window]   Fig. 3. Quantification by real-time RT-PCR of mRNA expression of various genes in single oocytes and embryos. Genotype (B6D2F1)-matched unfertilized MII oocytes, 2-cell IVF embryos, 2-cell cumulus clone embryos and 2-cell HSC clone embryos were analyzed. Dppa2, Dppa3, Dppa4, eIF-1A, ERV-L and Hdac1 are considered zygotically activated embryos according to previous studies. Except for Hdac3, values are expressed relative to those in the IVF group (value=1). For Hdac3, the values are expressed relative to Hprt values because of lack of Hdac3 expression in the IVF group. Values with different letters (a and b) differ significantly (P<0.05; Scheffe's F test).

View larger version (58K): [in a new window]   Fig. 4. Fluorescent images of 2-cell embryos stained with antibodies that recognized acetylated histones. Histone H3 acetylated on lysine 9 (H3K9) and H4K8, but not H3K14, are known to be sensitive to histone deacetylases. HSC clone embryos were more intensely stained for H3K9 and H3K14 than cumulus clone embryos or intracytoplasmic sperm injection (ICSI) embryos. Dot-like areas were frequently noted in HSC clone embryos (arrowheads). In HSC clone embryos, the two blastomeres were often stained differentially for H4K8. There was no difference in the staining pattern for H3K14 among the three types of embryos. 6-12 embryos were observed in each group.

View larger version (30K): [in a new window]   Fig. 5. Detection of donor-cell-specific gene expression in cloned embryos by RT-PCR. Transcription of donor-cell-specific genes was repressed in HSC clone and cumulus clone embryos. Each lane represents a single embryo, but results were confirmed by at least four replicates.

View larger version (29K): [in a new window]   Fig. 6. Real-time RT-PCR quantification of Hdac1 expression in donor HSCs, cumulus cells and other somatic cells freshly isolated from B6D2F1 female mice. Values are expressed relative to the Hprt expression level, which was assigned a value of 1.