Effect of cytoplast on the development of inter-subspecies nuclear transfer reconstructed goat embryo.

The aim of this study was to investigate effect of cytoplast on the development competence of reconstructed embryos derived from inter-subspecies somatic cell nucleus transfer (SCNT). First, the development potency of reconstructed embryos produced by transferring Boer goat fibroblast cell nucleus of different ages into enucleated Sannen goat ova was evaluated in order to determine which age of nuclear donor is favorable for the reconstructed embryos development. Secondly, the another component of reconstructed embryos, "cytoplast," was evaluated by comparing the effect of ovum cytoplast derived from Sannen male symbol x Boer female symbol descendant on the reconstructed embryos development to that of Sannen goat ovum cytoplast. The results revealed that the development rate of the reconstructed embryos derived from 2 months old Boer goat somatic cells was the highest, their gestation rate was up to 50%, and one viable male offspring was obtained. The cytoplast derived from the crossbreeding goats improves the development competence of reconstructed embryos, which birth rate was 5.5%. The genetic identification of offspring by using PCR-SSCP analysis confirmed that these cloned kids were derived from the donor. The results above reveal that the cytoplast of Sannen goat ovum could induce the dedifferentiation of somatic cell nuclei derived from Boer goat, but the reprogramming process of these reconstructed embryos seems incomplete, probably due to some incorrect processes happened after implantation. Relatedness components of nucleus donor in cytoplast of the crossbreeding goat may be helpful to induce the dedifferentiation of somatic cell nuclei completely and improve the development competence of the reconstructed embryos.

Study on Immune Efficacy of Single and Double Fusion DNA Vaccine from Mycobacterium bovis / 中国生物工程杂志

The DNA fragments of ag85b、esat-6、hsp65、mpb64 and ag85b-esat-6、hsp65-esat-6、mpb64-esat-6 were amplified by PCR and SOE technique.These seven fragments were inserted into pCDNA3.1(+)vector to construct recombinant plasmids pCA、pCE6、pCH、pCM、pCAE、pCHE and pCME.The seven plasmids were transfected into SP2/0 cell in vitro to detect the expression of target genes.BALB/c mice were intramuscularly vaccinated with the seven plasmids and the control vector pCDNA3.1(+)and PBS respectively.The serum antibodies and the spleen lymphocyte proliferation(SLP)and secreted IFN～? of spleen were tested.The results of indirect ELISA showed the levels of antibodies in all recombinant plasmids groups were significantly higher than the two control groups(P

Effect of human osteopontin on proliferation, transmigration and expression of MMP-2 and MMP-9 in osteosarcoma cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>To explore the effect of human osteopontin (hOPN) on the proliferation, transmigration and expression of matrix metallproteinase-2 (MMP-2) and matrix metallproteinase-9 (MMP-9) in osteosarcoma (OS) cells in vitro.</p><p><b>METHODS</b>The prokaryotic-expression vector of hOPN was produced. hOPN was then subcloned into E. coli BL21 (DE3) cells and purified with ProBond trade mark Columns. The proliferation, cell cycle and the expression of cyclin A in OS cells were investigated by using MTT assay, flow cytometry and Western blot respectively. The transmigration of OS cells was checked by using transwell cell culture chamber. The micro-pore-filter-membrane system was used to study the chemiotaxis of hOPN to OS cells. The levels of total protein were examined according to Coomassie Brilliant Blue manuals. The expression of MMP-2 and MMP-9 were evaluated by detecting the volume of degradation of gelatin on SDS-PAGE gel.</p><p><b>RESULTS</b>The prokaryotic-expression vector of hOPN and purified hOPN protein were achieved hOPN promoted OS cells proliferation in a dose-dependent manner, and stimulated cyclin A expression in OS cells to accelerate cell division cycle. hOPN facilitated the trans-membrane migration of OS cells. hOPN also enhanced the secretion of MMP-2 and MMP-9 in OS cells.</p><p><b>CONCLUSION</b>hOPN could stimulate cyclin A expression in OS cells. hOPN has chemiotaxis to OS cells and increases their transmigration. hOPN enhances the secretion of MMP-2 and MMP-9 in OS cells.</p>