Production of α1,3-galactosyltransferase targeted pigs using transcription activator-like effector nuclease-mediated genome editing technology

Recent developments in genome editing technology using meganucleases demonstrate an efficient method of producing gene edited pigs. In this study, we examined the effectiveness of the transcription activator-like effector nuclease (TALEN) system in generating specific mutations on the pig genome. Specific TALEN was designed to induce a double-strand break on exon 9 of the porcine α1,3-galactosyltransferase (GGTA1) gene as it is the main cause of hyperacute rejection after xenotransplantation. Human decay-accelerating factor (hDAF) gene, which can produce a complement inhibitor to protect cells from complement attack after xenotransplantation, was also integrated into the genome simultaneously. Plasmids coding for the TALEN pair and hDAF gene were transfected into porcine cells by electroporation to disrupt the porcine GGTA1 gene and express hDAF. The transfected cells were then sorted using a biotin-labeled IB4 lectin attached to magnetic beads to obtain GGTA1 deficient cells. As a result, we established GGTA1 knockout (KO) cell lines with biallelic modification (35.0%) and GGTA1 KO cell lines expressing hDAF (13.0%). When these cells were used for somatic cell nuclear transfer, we successfully obtained live GGTA1 KO pigs expressing hDAF. Our results demonstrate that TALEN-mediated genome editing is efficient and can be successfully used to generate gene edited pigs.

Methanolic Extract Isolated from Root of Lycoris aurea Inhibits Cancer Cell Growth and Endothelial Cell Tube Formation In Vitro

In this study, we investigated the effect of methanolic extract isolated from the root of Lycoris aurea (LA) on the growth of cancer cells and the tube formation activity of endothelial cells. Various cancer cells were treated with LA at doses of 0.3, 1, 3, 10 or 30 microg/ml and LA significantly suppressed the growth of several cancer cell lines, including ACHN, HCT-15, K-562, MCF-7, PC-3 and SK-OV-3, in a dose-dependent manner. We also found that LA induced cell cycle arrest at G2/M phase in ACHN renal cell adenocarcinoma cells. Further study demonstrated that LA concentration-dependently inhibited the tube formation, which is a widely used in vitro model of reorganization stage of angiogenesis, in human umbilical vein endothelial cells. Collectively, these results show that LA inhibits the growth of cancer cells and tube formation of endothelial cells and the growth-inhibitory effect of LA might be mediated, at least in part, by blocking cell cycle progression.

Noradrenergic Modulation of Spontaneous Inhibitory Postsynaptic Currents in the Hypothalamic Paraventricular Nucleus

Previous studies have suggested that brain stem noradrenergic inputs differentially modulate neurons in the paraventricular nucleus (PVN). Here, we compared the effects of norepinephrine (NE) on spontaneous GABAergic inhibitory postsynaptic currents (sIPSCs) in identified PVN neurons using slice patch technique. In 17 of 18 type I neurons, NE (30-100microM) reversibly decreased sIPSC frequency to 41+/-7% of the baseline value (4.4+/-0.8 Hz, p<0.001). This effect was blocked by yohimbine (2-20microM), an alpha2-adrenoceptor antagonist and mimicked by clonidine (50 microM), an alpha2-adrenoceptor agonist. In contrast, NE increased sIPSC frequency to 248+/-32% of the control (3.06+/-0.37 Hz, p<0.001) in 31 of 54 type II neurons, but decreased the frequency to 41+/-7% of the control (5.5+/-1.3 Hz) in the rest of type II neurons (p<0.001). In both types of PVN neurons, NE did not affect the mean amplitude and decay time constant of sIPSCs. In addition, membrane input resistance and amplitude of sIPSC of type I neurons were larger than those of type II neurons tested (1209 vs. 736 M omega, p<0.001; 110 vs. 81 pS, p<0.001). The results suggest that noradrenergic modulation of inhibitory synaptic transmission in the PVN decreases the neuronal excitability in most type I neurons via alpha2-adrenoceptor, however, either increases in about 60% or decreases in 40% of type II neurons.