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doi: 10.1242/10.1242/dev.00366

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Incomplete reactivation of Oct4-related genes in mouse embryos cloned from somatic nuclei

¹ Howard Hughes Medical Institute, 9 Cambridge Center, Cambridge, MA 02142, USA
² Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA
³ Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
⁴ Institute for Biogenesis Research and Department of Anatomy and Reproductive Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI 96822, USA

View larger version (50K): [in a new window]   Fig. 1. Expression of Oct4-related genes in somatic and embryonic tissues assayed by RT-PCR. Total cellular RNA samples were prepared from (left to right): adult brain, embryonic brain, embryonic limbs, adult skeletal muscles, lung, liver, spleen, kidney, heart, blastocysts (embryonic day E3.5), epiblasts (E7.0), purified primordial germ cells (E10), embryonic gonads (E12) and purified primitive type A spermatogonia (postnatal day 6). Gapd served as a ubiquitously expressed control. Oct4 is expressed in embryonic and germline tissues as described previously (Rosner et al., 1990; Yeom et al., 1996). Control reactions lacking reverse transcriptase or input cDNA showed no amplification products (data not shown). The ability of DNA-free reagent (Ambion) to eliminate residual genomic DNA in embryonic RNA samples was tested extensively in control experiments.

View larger version (56K): [in a new window]   Fig. 2. Gene expression during normal preimplantation development. Embryos of indicated developmental stages were harvested from pregnant females. Gene expression was assayed by RT-PCR in individual embryos. Gapd served as a ubiquitously expressed control. Expression of all test genes is readily detectable from the four-cell stage with the exception of Dppa1. Dppa1 expression is upregulated at the morula-to-blastocyst transition. Control reactions omitting cDNA or reverse transcriptase showed no amplification products (data not shown).

View larger version (73K): [in a new window]   Fig. 3. Gene expression in cumulus cells and in individual embryos cloned from cumulus cell nuclei. Cumulus cells were enzymatically separated from cumulus cell-oocyte complexes. Cumulus cells exhibit abundant expression of Hsh, Ptgs2 and Tnfsg6 genes but expression of these genes is not detectable in all cumulus clones regardless of their extent of development in vitro (Fulop et al., 1997a; Fulop et al., 1997b; Joyce et al., 2001). Ghost bands (marked with an asterisk) do not correspond to expected products and are presumed to be PCR artifacts. A small amount of Oct4, Dppa3, Dppa5 and Pramel7 expression detected in cumulus cell sample is likely to be the result of contamination by mRNAs from oocytes damaged during the isolation procedure. Oct4 expression is clearly limited to the oocyte in the follicle (Pesce et al., 1998). Pre-morula pm8, morula m2 and blastocysts b4 and b14 are examples of incompletely reprogrammed cumulus clones. A full summary of gene expression is shown in Fig. 4.

View larger version (18K): [in a new window]   Fig. 4. Summary of embryonic gene expression in all studied clones. Cloned embryos are named according to the developmental stage to which they progressed during 4 days of culture following nuclear transfer (pm, pre-morula embryos; m, morula; b, blastocyst). Investigated genes are shown in columns with gray squares indicating expression and white squares lack of expression of a given marker gene. Embryos within each developmental group are arranged such that the most transcriptionally defective are shown above those with fewer defects. Embryos with identical expression profiles are combined in a single row. Some of the primary data are shown in Figs 3 and 5.

View larger version (59K): [in a new window]   Fig. 5. Gene expression in ES cells and in individual cloned embryos from ES cell nuclei. Oct4 and Oct4-related genes are abundantly expressed in the donor ES cells and in ES cell nuclei-derived clones. Note that although Dppa1 gene is strongly expressed in ES cells, it is initially downregulated in ES cell cloned morulae before re-activation in the blastocyst stage ES clones. This pattern of Dppa1 expression is reminiscent of its expression during normal embryogenesis (see Fig. 2). A full summary of gene expression is shown in Fig. 4.