Xiaoning He

An immortalized goat mammary epithelial cell line induced with human telomerase reverse transcriptase (hTERT) gene transfer

Although mammary epithelial cell lines can provide a rapid and reliable indicator of gene expression efficiency of transgenic animals, their short lifespan greatly limits this application. To provide stable and long lifespan cells, goat mammary epithelial cells (GMECs) were transduced with pLNCX2-hTERT by retrovirus-mediated gene transfer. Transduced GMECs were evaluated by reverse transcriptase polymerase chain reaction (RT-PCR), proliferation assays, karyotype analysis, telomerase activity assay, western blotting, soft agar assay, and injection into nude mice. Non-transduced GMECs were used as a control. The hTERT-GMECs had higher telomerase activity and extended proliferative lifespan compared to non-transfected GMECs; even after Passage 50, hTERT-GMECs had a near diploid complement of chromosomes. Furthermore, they did not gain the anchorage-independent growth property and were not associated with a malignant phenotype in vitro or in vivo.

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.

Ipr1 Gene Mediates RAW 264.7 Macrophage Cell Line Resistance to Mycobacterium bovis

Tuberculosis caused by Mycobacterium bovis (M. bovis) seriously affects efficiency of animal production with impacts on public health as well. Effective programmes of prevention and eradication of M. bovis infection therefore are urgently needed. Intracellular pathogen resistance gene 1 (Ipr1) is well known to mediate innate immunity to Mycobacterium tuberculosis (MTB), but there are no reports as to whether Ipr1 can enhance the phagocytic ability of macrophage against M. bovis. In this investigation, RAW 264.7 macrophage was transduced with lentiviral vector carrying Ipr1 (named Lenti‐Ipr1); transgenic cells were identified by RT‐PCR and western blotting. Transgenic positive cells (R‐Ipr1) were then infected with an M. bovis virulent strain, with non‐transduced cells used as control. When cell proliferation, viability and apoptosis of the two groups were investigated, it was found that infected RAW 264.7 died by necrosis whereas R‐Ipr1 underwent apoptosis. Furthermore, the numbers of intracellular bacteria in R‐Ipr1 were lower than those in control cells (P < 0.05). To identify the role of Ipr1, we measured the genes of Casp3, Mcl‐1 and NOS2A which associated with macrophage activation and apoptosis by real‐time quantitative PCR. The results demonstrated that Ipr1 gene expression can enhance anti‐M. bovis infection of macrophage. This establishes a basis for the future production of Ipr1‐transgenic cattle to strengthen the tuberculosis resistance.

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.

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.

Construction of Ipr1 expression vector and development of cloned embryos in vitro.

The purpose of this study was to prepare intracellular pathogen resistance 1 (Ipr1) transgenic donor cells for somatic cell nuclear transfer (SCNT). Based on our current understanding of Ipr1, a macrophage special expression vector pSP-EGFP-Ipr1was constructed. Bovine fetal fibroblasts were transfected with pSP-EGFP-Ipr1. The green fluorescent protein (GFP)-expressing cells were selected and transferred into enucleated bovine oocytes. Then, the rates of oocyte cleavage and blastocyst formation of transgenic cells and non-transgenic cells were observed, respectively. The results showed that reconstructed embryos derived from transgenic cells could successfully develop into blastocysts, most of which were GFP-positive. This study may provide cloned embryos for the production of anti-tuberculosis transgenic animals.