Recombinant adenovirus-mediated human telomerase reverse transcriptase gene can stimulate cell proliferation and maintain primitive characteristics in bovine mammary gland epithelial cells.

The human telomerase reverse transcriptase (hTERT) gene has been used to stimulate the proliferation of most types of human cells. The present study was designed to evaluate the feasibility and efficiency of adenovirus-mediated hTERT in the proliferation of bovine mammary gland epithelial cells (bMGEs). A plasmid and an adenovirus vector that carried hTERT, namely pEGFP- hTERT and Ad- hTERT, were constructed and transfected into bMGEs, respectively. In order to select the best strategy for stimulating cell proliferation, the adenovirus- and plasmid-mediated hTERT were compared in terms of the positive cloning and transgenic efficiency. The results showed that only Ad- hTERT had high infection efficiency and produced a positive polyclone population (hTERT-bMGEs). The characteristics of the hTERT-bMGEs were investigated with further analysis by reverse transcription-polymerase chain reaction (RT-PCR), western blotting, proliferation assays, and flow cytometry, which showed that hTERT facilitated strong cell proliferation. Real-time quantitative PCR showed a normal level of expression of beta-casein, the caspase-8 and c-myc proto-oncogene, and immunofluorescence demonstrated the properties of the epithelial cells. In conclusion, the adenovirus-mediated hTERT gene could not only extend the cell lifespan, but also maintained the primary characteristics of the cells. It may be possible to extend the use of a wide variety of non-human mammalian cells in this way. This study has provided additional insight into the mechanism of cell proliferation by demonstrating the lack of integration of the adenovirus-mediated hTERT gene into the mammalian genome.

Adipogenic differentiation and EGFP gene transfection of amniotic fluid-derived stem cells from goat fetus at terminal gestational age.

The aims of this study were to determine whether stem cells could be isolated from amniotic fluid of goat fetus at terminal gestational age and to determine if these stem cells could differentiate into adipogenic cells and be transfected with a reporter gene, EGFP (enhanced green fluorescent protein). The stem cells were isolated from amniotic fluid of goat fetus at terminal gestational age, induced to differentiate into adipogenic cells in vitro and transfected with the EGFP gene using lipofection. Markers associated with undifferentiated AFS (amniotic fluid-derived stem) cells were tested by RT (reverse transcription)-PCR. The results demonstrated that AFS cells could be isolated from amniotic fluid of goat fetus at terminal gestational age and could differentiate into adipogenic cells. The EGFP gene was transfected into AFS cells successfully. EGFP gene transfection efficiency of the three groups of transgenic AFS cells were 26.0, 29.9 and 30.5%, respectively. Both transgenic and wild-type AFS cells could express Hes1 (hairy and enhancer of split 1), Oct4 (octamer-binding protein 4) and Nanog.

Multipotent differentiation of the EGFP gene transgenic stem cells derived from amniotic fluid of goat at terminal gestational age.

We have isolated stem cells from amniotic fluid of goat at terminal gestational age and transferred the EGFP (enhanced green fluorescent protein) gene into the stem cells previously. The aim of this study was to determine whether the transgenic stem cells have the capability of multipotent differentiation. The transgenic stem cells were induced to differentiate into neurogenic, adipogenic, osteogenic and endothelial cells in vitro. Markers associated with AFS (amniotic fluid-derived stem) cells and the differentiated cells were tested by RT-PCR (reverse transcription-PCR). The results demonstrated that the transgenic AFS cells were capable of self-renewal, a defining property of stem cells. AFS cells were positive for the undifferentiated cell markers, Oct4, Nanog, Sox2 and Hes1, while following differentiation cells expressed markers for neurogenic cells such as astrocyte [GFAP (glial fibrillary acidic protein)] and NSE (neuron-specific enolase), adipogenic cells [LPL+ (lipoprotein lipase+)], osteogenic cells (osteocalcin+ and osteonectin+) and endothelium [CD34+ and eNOS+ (endothelial nitric oxide synthase)]. The results demonstrated that the EGFP gene transgenic AFS cells have the capability of multipotent differentiation, which means that the transgenic AFS cells may be useful in cell-transplantation studies in future.

Differentiation and characteristics of the enhanced green fluorescent protein gene transgenic goat neural stem cells cultured in attached and non-attached plates.

The aims of this study were (i) to determine whether NSCs (neural stem cells) could be isolated from the brain of embryonic day 98 fetal goat, (ii) to determine if these stem cells have the capability of multipotent differentiation following transfection with a reporter gene, EGFP (enhanced green fluorescent protein) and (iii) to study the characteristics of the stem cells cultured in attached and non-attached plates. NSCs were isolated from embryonic day 98 fetal goat brain, transfected with EGFP gene using lipofection, and subcultured in attached and non-attached plates respectively. The transgenic stem cells were induced to differentiate into osteogenic and endothelial cells in vitro respectively. Markers associated with undifferentiated NSCs and their differentiated cells were tested by RT-PCR (reverse transcription-PCR). The results demonstrated that stem cells could be isolated from embryonic day 98 fetal goat brain, and EGFP gene could be transfected into the cells. The transgenic NSCs were capable of self-renewal, a defining property of stem cells, and were grown as free-floating neurospheres in non-attached plates. When the neurospheres were transferred and cultured in attached plates, cells migrate from the neurospheres and are grown as spindle cells. The stem cells were grown as quasi-circular cells when the single stem cells were cultured in attached plates. Both the NSCs cultured in non-attached and attached plates could express Hes1 (hairy and enhancer of split 1), Oct4 (octamer-binding protein 4), Nanog, Sox2 [SRY (sex-determining region Y)-box 2] and Nestin, while following differentiation cells expressed markers for osteogenic cells (Osteocalcin+ and Osteonectin+) and endothelium (CD34+ and eNOS+). The results demonstrated that the goat EGFP gene transgenic NSCs have the capability of multipotent differentiation, which means that the transgenic NSCs may be useful in cell transplantation studies in future.

Differentiation of AFS cells derived from the EGFP gene transgenic porcine fetuses.

We have obtained the EGFP (enhanced green fluorescence protein) gene transgenic porcine fetuses before. The aims of this study were (i) to determine whether stem cells could be isolated from amniotic fluid of the transgenic porcine fetuses, and (ii) to determine if these stem cells could express EGFP and differentiate in vitro. The results demonstrated that stem cells could be isolated from amniotic fluid of the EGFP gene transgenic porcine fetuses and could express EGFP and differentiate in vitro. Undifferentiated AFSs (amniotic fluid-derived stem cells) expressed POU5F1, THY1 and SOX2, while the following differentiation cells expressed markers for chondrogenic (COL2A1), osteogenic (osteocalcin and osteonectin) and neurogenic cells such as astrocyte (GFAP), oligodendrocyte (GALC) and neuron (NF, ENO2 and MAP).

Osteogenic and neurogenic differentiation of EGFP gene transfected neural stem cells derived from the brain of porcine fetuses at intermediate and late gestational age.

The aims of this study were (i) to determine whether NSCs (neural stem cells) could be isolated from the brain of porcine fetuses at intermediate and late gestational age and (ii) to determine if these stem cells could be differentiated in vitro into osteogenic and neurogenic lineages following transfection with a reporter gene, EGFP (enhanced green fluorescence protein). The NSCs were isolated from the brains of porcine fetuses at intermediate and late gestational age and transfected with EGFP gene using lipofection. The transfected NSCs cells were induced to differentiate into cells of osteogenic and neurogenic lineages. Markers associated with NSCs and their osteogenic and neurogenic derivatives were tested by PCR. The results demonstrated that NSCs could be isolated from the brain of porcine fetus at intermediate and late gestational age and that transfected NSCs expressed EGFP and could be induced to differentiate in vitro. NSCs expressed CD-90, Hes1, Oct4, Sox2 and Nestin, while following differentiation cells expressed markers for osteogenic (osteocalcin and osteonectin) and neurogenic cells such as astrocyte [GFAP (glial fibrillary acidic protein)], oligodendrocyte [GALC (galactosylceramide)] and neuron [NF (neurofilament), ENO2 (enolase 2) and MAP (microtubule-associated protein)].

Development of cloned embryos from porcine neural stem cells and amniotic fluid-derived stem cells transfected with enhanced green fluorescence protein gene.

We assessed the developmental ability of embryos cloned from porcine neural stem (NS) cells, amniotic fluid-derived stem (AFS) cells, fetal fibroblast cells, adult fibroblast, and mammary gland epithelial cells. The five cell lines were transfected with enhanced green fluorescence protein gene respectively using lipofection. NS and AFS cells were induced to differentiate in vitro. Stem cells and their differentiated cells were harvested for analysis of the markers using RT-PCR. The five cell lines were used for nuclear transfer. The two-cell stage-cloned embryos derived from each cell line were transferred into the oviducts of surrogate mothers. The results showed that both NS and AFS cells expressed POU5F1, THY1 and SOX2, and they were both induced to differentiate into astrocyte (GFAP+), oligodendrocyte (GalC+), neuron (NF+, ENO2+, and MAP2+), adipocyte (LPL+ and PPARG-D+), osteoblast (osteonectin+ and osteocalcin+), myocyte (MYF6+ and MYOD+), and endothelium (PECAM1+, CD34+, CDH5+, and NOS3+) respectively. Seven cloned fetuses (28 days and 32 days) derived from stem cells were obtained. The in vitro developmental ability (morula-blastocyst rate was 28.26-30.07%) and in vivo developmental ability (pregnancy rate were 1.67-2.17%) of the embryos cloned from stem cells were higher (P<0.05) than that of the embryos cloned from somatic cells (morula-blastocyst rate was 16.27-19.28% and pregnancy rate was 0.00%), which suggests that the undifferentiated state of the donor cells increases cloning efficiency.

[Nuclear transfer and mitochondria].

Mitochondria, which can produce and supply energy for mammals, are involved in many cellular events of growth, development, aging, apoptosis as well as diseases. Nuclear transfer could result in mitochondria heteroplasmy in cloned embryos and offspring, affecting the phenotypes of the individuals and even causing mitochondrial diseases. This text has expounded the biological functions and the hereditary characteristics of the mitochondria in mammals, and analyzed the change of donor and recipient mitochondria in embryos and offspring derived from intraspecific and interspecific embryonic or somatic nuclear transfer as well as several factors which might influence mitochondrial heteroplasmy in the process of nuclear transfer. The mitochondrial diseases it may cause and their solutions are also briefly presented.

Improved development of somatic cell cloned bovine embryos by a mammary gland epithelia cells in vitro model.

Previous studies have established a bovine mammary gland epithelia cells in vitro model by the adenovirus-mediated telomerase (hTERT-bMGEs). The present study was conducted to confirm whether hTERT-bMGEs were effective target cells to improve the efficiency of transgenic expression and somatic cell nuclear transfer (SCNT). To accomplish this, a mammary-specific vector encoding human lysozyme and green fluorescent protein was used to verify the transgenic efficiency of hTERT-bMGEs, and untreated bovine mammary gland epithelial cells (bMGEs) were used as a control group. The results showed that the hTERT-bMGEs group had much higher transgenic efficiency and protein expression than the bMGEs group. Furthermore, the nontransgenic and transgenic hTERT-bMGEs were used as donor cells to evaluate the efficiency of SCNT. There were no significant differences in rates of cleavage or blastocysts or hatched blastocysts of cloned embryos from nontransgenic hTERT-bMGEs at passage 18 and 28 groups (82.8% vs. 81.9%, 28.6% vs. 24.8%, 58.6% vs. 55.3%, respectively) and the transgenic group (80.8%, 26.5% and 53.4%); however, they were significantly higher than the bMGEs group (71.2%, 12.8% and 14.8%), (p < 0.05). We confirmed that hTERT-bMGEs could serve as effective target cells for improving development of somatic cell cloned cattle embryos.

Inducible expression of enhanced green fluorescent protein by interleukin-1α, interleukin-1ß and Toll-like receptor 2 promoters in goat mammary epithelial cells in response to bacterial challenges.

The development of a bacteria-inducible expression system has several advantages compared with persistent expression of anti-bacterial proteins in milk to prevent and treat mastitis. The present study determined whether mastitis responsive promoters could regulate enhanced green fluorescent protein (EGFP) expression in goat mammary epithelial cells (GMECs) in response to challenges with Escherichia coli, Staphylococcus aureus or Streptococcus agalactiae. The level of expression of interleukin (IL)-1α was significantly increased in GMECs challenged with E. coli, S. aureus or S. agalactiae compared with untreated GMECs. IL-1ß was induced by E. coli and S. aureus, while Toll-like receptor 2 (TLR2) was induced by E. coli only. GMECs were transfected with IL-1α, IL-1ß and TLR2 promoter-EGFP reporter gene lentiviral expression vectors and the levels of expression of EGFP were measured by flow cytometry and Western blot analysis after bacterial challenge. EGFP expression driven by the IL-1α and IL-1ß promoters was higher in GMECs challenged with E. coli, S. aureus or S. agalactiae than in untreated GMECs. There were no differences in EGFP expression driven by the TLR2 promoter between GMECs challenged with S. aureus or S. agalactiae and untreated GMECs, but EGFP expression was significantly increased in GMECs challenged with E. coli. Overall, these results indicate that the promoters of some bacteria-inducible genes can regulate EGFP expression in GMECs in response to bacterial challenges. This bacteria-inducible expression strategy could be used for production of mastitis resistant animals by regulating the expression of anti-bacterial proteins in the mammary gland.

[Demecolcine-induced enucleation of Kunming mouse oocyte].

On the basis of exiting technique pathway of demecolcine-induced enucleation(IE), several factors (Demecolcine concentration, time of demecolcine inition and treatment, oocytes age) affecting the IE rate were tested using Kunming mouse oocytes. The experiments' results demonstrated that: In experiment 1,activated oocytes could be enucleated efficiently by treating with KSOM medium containing 0.4 microg/mL or 0.5 microg/mL demecolcine for 60 min, but 0.5 microg/mL group gained the higher IE rate(33.3%). In experiment 2, maximum IE rate (31.9% -approximately 24.5%) were obtained when oocytes were exposed to 0.5 microg/mL demecolcine between 0 and 5 min postactivation and treated for 60 -approximately 180 min. In experiment 3,oocytes collected from Kunming mouse at 17 -approximately 18h after hCG administration were favoriate to demecolcine-IE(27.1%). By comparison and analysis of the data, we established the optimized IE procedure.

MicroRNA-34c expression in donor cells influences the early development of somatic cell nuclear transfer bovine embryos.

The essence of the reprogramming activity of somatic cell nuclear transfer (SCNT) embryos is to produce normal fertilized embryos. However, reprogramming of somatic cells is not as efficient as the reprogramming of sperm. In this report, we describe the effect of an inducible, specific miR-34 microRNA expression in donor cells that enables a similar level of sperm:transgene expression on the early development of SCNT embryos. Our results showed that donor cells with doxycycline (dox)-induced miR-34c expression for the preparation of SCNT embryos resulted in altered developmental rates, histone modification (H3K9ac and H3K4me3), and extent of apoptosis. The cleavage rate and blastocyst formation of the induced nuclear transfer (NT) group were significantly increased. The immunofluorescence signal of H3K9ac in embryos in the induced NT group significantly increased in two-cell- and eight-cell-stage embryos; that of H3K4me3 increased significantly in eight-cell-stage embryos. Although significant differences in staining signals of apoptosis were not detected between groups, lower apoptosis levels were observed in the induced NT group. In conclusion, miR-34c expression induced by dox treatment enhances the developmental potential of SCNT embryos, modifies the epigenetic status, and changes blastocyst quality.

Site-directed mutagenesis of the myostatin gene in ovine fetal myoblast cells in vitro.

Myostatin is an important negative regulator of muscle growth and development. Natural mutations of the myostatin gene cause a double muscling phenotype in beef cattle, pigs and sheep. Therefore, it is feasible to produce a high growth domestic breed by generating a transgenic animal with a mutation, deletion or knockout of the myostatin gene. Our objective was to introduce a subtle mutation of G to A 281-bp upstream of the 3' untranslated region (3'UTR) end of the myostatin gene in Poll Dorset fetal myoblast cells in vitro. Fetal myoblast cells were isolated from fetuses at day 50 of gestation from Poll Dorset sheep and transfected with linear gene-targeting vector pMSTN-A using electroporation. We obtained seven gene-targeted cell colonies with homologous recombination, which were positive as confirmed by PCR, Southern blot. The Western blot analysis result demonstrated that the myostatin protein expression in positive colonies is lower than that of negative ones. These results strongly suggest that we successfully mutated the myostatin gene of Poll Dorset ovine fetal myoblast cells and the mutation can effectively downregulate the myostatin protein expression.

Characteristics and EGFP expression of porcine mammary gland epithelial cells.

The aims of this study were to establish a porcine mammary gland epithelial (PMGE) cell line, and to determine if these PMGE cells could be maintained long-term in culture by continuous subculturing following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Primary culture of PMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating pig. The passage sixteen PMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in PMGE cells was tested by immunofluorescence. Βeta-Casein gene mRNA was tested for PMGE cells by RT-PCR. The results showed that PMGE cells could form dome-like structure which looked like nipple, and the cells contained different cell types. The expression of Cell keratins demonstrated the property of epithelial cells, and the PMGE cells could express transcript encoding a Βeta-Casein protein. EGFP gene was successfully transferred into the PMGE cells, and the transfected cells could be maintained long-term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected porcine mammary gland epithelial (ET-PMGE) cell line.

Comparation of enhanced green fluorescent protein gene transfected and wild-type porcine neural stem cells.

The aim of this study was to transfect and express the enhanced green fluorescence protein (EGFP) gene into porcine neural stem cells (NSCs) to determine whether EGFP can be used as a marker to monitor NSCs. NSCs were isolated from embryonic day 30 fetal pig brain and transfected with EGFP gene using lipofection. Transfected and wild-type NSCs were induced to differentiate into cells of neuronal and myogenic lineages. Markers of passage three NSCs and their differentiated cells were tested by reverse transcription polymerase chain reaction. The results showed that EGFP could be expressed in NSCs and the differentiated cells. NSCs expressed Nestin, NogoA, DCX, Hes1, Oct4, CD-90 and Sox2. NSCs could differentiated into astrocyte (GFAP(+)), oligodendrocyte (GalC(+)), neuron (NF(+), NSE(+) and MAP2(+)) and myocyte (myf-6(+) and myoD(+)). We concluded that EGFP can be used as a marker in monitoring NSCs.

[Somatic nuclear transplantation and serial nuclear transplantation of human finger-domain lacking t-PA gene in goat].

In order to research developmental competence of transgenic somatic cell by serial nuclear transplantation, goat cloned embryos were compared with recloned embryos in ability of in vitro development. Fetal fibroblasts including human finger-domain lacking t-PA gene was microinjected into cytoplasm of the MII oocytes. Goat embryos (G0) were cloned by this procedure. A single blastomere from 16 - 64-cell goat cloned embryos (G0) was microinjected into Intracytoplasm of the MII oocytes. Goat embryos (G) were cloned by this procedure. Goat embryos (G2, G3) were recloned by using 16 - 64-cell recloned embryos. The developmental time of donor embryo affected the developmental rate of recloned embryos (G1, G2). The results show: the cleavage rate of cloned embryos (G0) (76.45% +/- 1.17%) was no difference significantly with recloned embryos (G1 G2 G3) (72.18% +/- 1.97%, 76.05% +/- 2.38%, 75.99% +/- 2.84%); the developmental rate of morulae and blastocysts of cloned embryos (47.20% +/- 2.93%, 11.00% +/- 1.42%) were higher than these of recloned embryos(34.99% +/- 2.66%, 28.23% +/- 2.00%, 23.34% +/- 1.99%) (3.87% +/- 0.67%, 2.08% +/- 1.66%, 0); the morulae rate(29.57% +/- 1.53%, 24.43% +/- 1.87%) and blastocysts rate(1.96% +/- 1.31%, 2.01% +/- 1.34%) of recloned embryos (G1 G2) from 16-cell recloned embryos were lower than those(34.32% +/- 1.31%, 29.76% +/- 1.66% and 3.86% +/- 1.03%, 3.48% +/- 0.34% )from 32 - 64-cell recloned embryos (P > 0.05). In conclusion, nuclear transfer embryos should not were recloned mostly; and the embryos recloned by using 32 - 64-cell embryos achieved higher developmental ability compared with using 16-cell embryos.