Generation of α-1,3-Galactosyltransferase-Deficient Porcine Embryonic Fibroblasts by CRISPR/Cas9-Mediated Knock-in of a Small Mutated Sequence and a Targeted Toxin-Based Selection System.

The CRISPR/Cas9 system has enabled the editing of mammalian genomes; however, its applicability and efficiency in the pig genome has not been studied in depth. The α-gal epitope synthesized by α-1,3-galactosyltransferase gene (GGTA1) is known as a xenoantigen obtained upon pig-to-human xenotransplantation. We here employed the CRISPR/Cas9 system-mediated knock-in of endogenous GGTA1 via targeted homologous recombination (HR). Linearized donors with ~800-bp homology flanking the CRISPR/Cas9 target site [exon 4 (containing ATG) of GGTA1] served as a template for gene targeting by HR. Using a targeted toxin strategy to select clones lacking α-gal epitope expression, we successfully obtained several knock-in clones within 3 weeks of initial transfection. These results suggest that the use of CRISPR/Cas9-mediated HR to knock-in a mutated fragment at defined loci represents an efficient strategy to achieve the rapid modulation of genes of interest in swine cells and is a promising tool for the creation of KO piglets.

Inhibitory factor of actomyosin ATPase in the cardiac conduction system.

ATPase activation and superprecipitation of natural actomyosin in the cardiac conduction system (specialized myocardium) and ventricle were compared. The ATPase activity and superprecipitation of natural actomyosin from the specialized myocardium were much lower than those from the ventricle. The sensitivity of natural actomyosin ATPase to calcium was higher in the specialized myocardium than in the ventricle, whereas the maximum activity of the specialized myocardium was lower than that of the ventricle. The Ca2+-desensitized actomyosin of the specialized myocardium presented scarcely any superprecipitation. Addition of the 0-40% ammonium sulfate fraction (mainly containing myosin and actin) of natural actomyosin from the specialized myocardium to ventricular natural actomyosin inhibited superprecipitation of the latter. On the other hand, addition of the 40-60% ammonium sulfate fraction (mainly containing troponins and tropomyosin) of natural actomyosin from the specialized myocardium enhanced superprecipitation of ventricular natural actomyosin. These results suggested that the specialized myocardium contains some factor(s) that inhibits actin-myosin interaction.

Distribution and metabolism of norepinephrine, cyclic AMP and cyclic GMP in the atrioventricular conducting tissue of the bovine heart.

The distribution of norepinephrine (NE), cyclic AMP (cAMP) and cyclic GMP (cGMP) and the activities of related enzymes in the atrioventricular (A-V) conducting tissue of the bovine heart were examined. The concentration of NE in the atrium was about twice that in the ventricle. In the A-V conducting tissue, the concentration of NE was highest in the atrioventricular node (AVN) and lowest in the false tendon (FT), with intermediate levels in the bundle of His (HIS) and the right and left bundle branches (RLBB). The activity of monoamine oxidase (MAO) in the atrium was about 2.2 times that in the ventricle. In the A-V conducting tissue, the activity of MAO was highest in the HIS and lowest in the FT. The activity of catechol-o-methyltransferase (COMT) in the atrium and ventricle was similar, and that in the HIS was slightly, but not significantly, higher than that in other regions of the A-V conducting tissue. The concentration of cAMP in the ventricle was about twice that in the atrium. In the A-V conducting tissue, the concentration of cAMP was higher in the AVN and FT than in the HIS and RLBB. The distribution of adenylate cyclase (AC) was similar to that of NE. The phosphodiesterase (PDE) activity in the atrium and ventricle was similar. No significant difference was found in the level of PDE activity in different regions of the A-V conducting tissue. The concentration of cGMP was slightly, but not significantly, higher in the A-V conducting tissue than in the atrium or ventricle. In the A-V conducting tissue, the concentration of cGMP was highest in the FT and the concentrations in the HIS, RLBB and AVN were similar. These findings suggest that in the A-V conduction tissue, the regions that have the higher spontaneous pacemaker rates have higher NE content and AC activity, that is sensitivity to NE. Furthermore, the sensitivity for muscarinic cholinergic stimulation is higher in the conducting tissue (especially in the FT) than in the atrium and ventricle.