| Embryonic stem (ES) cell lines provide an invaluable research tool for genetic engineering, developmentalbiology and disease models. These cells can be maintained indefinitely in culture and yet maintain competence to produce all the cells within a fetus. Domesticated ungulate ES cell lines would also be useful in the precise genetic engineering of these animals for improved production traits and products, for disease resistance and for biopharming. While mouse ES cell lines were first established over two decades ago and primate ES cells in the 1990s, validated ES cell lines have yet to be established in ungulates.The aim of this study was to establish Nanog-expressing cell lines that can be used as donor cells for nuclear transfer in cattle, and forther to find a new approach to derivation of bovine ES cell lines. The important results were listed below.(1) By reverse transcription–polymerase chain reaction (RT-PCR), the complete cDNA of Nanog gene was cloned from fetal bovine primordial genital ridge cells at six weeks of age. Confirmed by DNA sequencing, the fragment was composed of 903 nucleotides, and shared 99.9% identity with bovine Nanog CDS, only one nucleotide substitution—170 (T to C), concomitant with one amino acid substitution—58 (Thr to Ile,).(2) Prokaryotic expression vector of bovine Nanog gene was successfully constructed. the recombinant plasmid pGEX-KG -Nanog was transformed and induced by IPTG to express GST- Nanog fusion protein in JM109. As indicated by western blotting , GST-Nanog fusion protein expressed efficiently in JM109 and reactivated with GST antibody, as what we desired, the fusion protein is about 60KD, which can be used to prepare polyclonal antibody of bovine Nanog gene.(3) Eukaryotic expression vectors—pEGFP-Nanog and pFLAG-Nanog were also successfully constructed . Both of the recombinant plasmids were transfected into skin fibroblast cells. Stable transfected cell lines, 3 for pEGFP-Nanog and 1 for pFLAG-Nanog, were successfully established after two months of selection with neomycine (G418) and proliferation.(4) RT-PCR and Western Blotting assays showed that Nanog mRNA and EGFP-Nanog(60KD)or FLAG-Nanog(34KD) fusion protein were expressed in these cell lines, indicating that Nanog gene have been expressed in bovine fibroblast cells.(5) Immunohistochemistry or fluorescence immuno-staining showed that, as well as Nanog , the other two stem cell markers, Oct-4 and SSEA-4, were also expressed in these Nanog-expressing fibroblast cell lines.(6) Flowcytometry analysis of stem cell superfacial markers illustrated that the positive rate of CD71,CD29 and CD11a in FLAG-Nanog-expressing fibroblast cells increased by 67%,2.5% and 7% respectively than those in intact fibroblast cells(BEF422).(7) After culture in suspension, the FLAG-Nanog-expressing fibroblast cells formed typical embroid bodies, and RT-PCR detection showed that molecular markers of 3 germ layers were expressed in the embroid bodies.(8) The pluripotency assays above indicated that the Nanog-expressing cells were to some extent pluripotent.(9) The EGFP-Nanog/ FLAG-Nanog expressing fibroblast cells and intact fibroblast cells(BEF422) were respectively used to construct cloned embryos. The results showed that , although the cleavage rate of recombinant embryos in BEF422 was significantly (P<0.05) higher than in EGFP-Nanog expressing cells (82.14% vs 40.38 % and 52.94.0% ), but there was no significant difference in the development rates to blastocysts on day 7 to 9 between them (14.29% vs 12.82% and11.76% ) (P>0.05), indicating that after cleavage the transgenic cloned embryos had the same development competence as the untransgenic ones with normal somatic cells..
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