[Heterogenous expression and enzymatic property analysis of microbial-derived pectinases in pig PK15 cells].

As one of plant cell wall components, pectin is the main anti-nutritional factor in livestock and poultry feeds and has an adverse effect on utilization efficiency of feed energy and nitrogen. Pectinases, which are widely found in microorganisms such as bacteria, yeast and filamentous fungi in nature,can improve feed efficiency by relieving the anti-nutritional effect of pectin through promoting the hydrolysis reaction of feed pectin. To explore the feasibility of expressing microbial-derived pectinase genes in pig cells, we introduced microbial-derived pectinase genes pg5a, pgI, pga3A, and pgaA into porcine PK 15 cells by lipofection for heterogenous expression. Enzymatic activities of the pectinases encoded by these genes were analyzed using the 3,5 dinitrosalicylic acid (DNS) method. Results showed that all four pectinase genes were able to be transcribed into mRNAs in porcine PK 15 cells, but only pg5a and pgI were adapted to the porcine cell expression system. Among them, the maximum activity of pectinase PG5A was 0.95 U/mL, the optimum pH was pH 4.0, and the enzymatic activity was maintained above 46% in the range of pH 4.6 to 6.0. Pectinase PGI obtained the highest enzymatic activity at pH 5.0, which was 0.30 U/mL, and maintained more than 35% of the activity in the range of pH 4.0 to 6.0. The results of digestive protease tolerance test showed that PG5A and PGI were highly resistant to pepsin and trypsin. After treatment with 1 mg/mL pig pepsin for two hours, the residual enzymatic activities of PG5A and PGI were 76% and 71%, respectively. And after two hours treatment with 1 mg/mL of pig trypsin, the remaining enzymatic activities of PG5A and PGI were 44% and 93%, respectively. In summary, pectinase PG5A and PGI can be effectively expressed in pig cells, and have strong tolerance to pig intestinal pH environment and digestive proteases. Therefore, both pg5a and pgI can be used as candidate genes for production of transgenic pigs.

[MEK inhibitor PD0325901 significantly boosts ssODN-mediated HDR efficiency in porcine fetal fibroblasts].

There are two major pathways, homology-directed repair (HDR) and nonhomologous end joining (NHEJ), involved in double-strand break (DSB) repair. Single-stranded oligodeoxyribonucleotide (ssODN)-mediated homologous recombination repair is commonly used for animal site-directed genome editing, with great scientific and practical value. To improve ssODN-mediated HDR efficiency in the pig genome, we investigated the effect and molecular mechanism of mitogen-activated extracellular signal-regulated kinase (MEK) inhibitor PD0325901 on the HDR efficiency in porcine fetal fibroblasts (PFFs). The results showed that PD0325901 obviously increased the percentage of G2 and S phase cell populations and reduced the cell population ratio in the G1 phase of PFFs, and promoted the expression of HDR repair factor. At the optimal concentration of 250 nmol/L, PD0325901 increased the repair efficiency of ssODN-mediated GFP reporter vector by 58.8% and the directed editing efficiency of PFF DMD and ROSA26 locus by 48.16% and 17.64%, respectively. The results show that MEK inhibitor PD0325901 significantly promotes the efficiency of ssODN-mediated homologous-directed repair in the porcine genome, thus offering a new idea to generate genetically modified pigs more effectively.

Effects of parameters, plasmid dosages and topological structures on transfection efficiency of porcine fetal fibroblasts using different electroporators.

To obtain an ideal transfection efficiency of porcine fetal fibroblasts, fluorescence activated cell sorting (FACS) was used to optimize parameters for transfection of porcine fetal fibroblasts (PFFs) with ECM? 830, NEPA 21 and Nucleofector? 2b in different conditions such as electroporation parameters, plasmid dosages and topological structures. The results show that the optimum poring pulse parameter of NEPA 21 is voltage 200 V, continuous 3 ms, interval 50 ms, 3 times, voltage attenuation range of 10%; and the transfection efficiency of Nucleofector? 2b is highest under U-023 program. Under the optimum conditions, FACS analysis demonstrates that Nucleofector? 2b and ECM? 830 have the highest transfection efficiency when transfecting 10 µg supercoiled plasmids into PFFs, and 8 µg for NEPA 21. Supercoiled plasmids show higher transfection efficiencies than linearized plasmids. Moreover, Nucleofector? 2b has the highest transfection efficiency among the three electroporation instruments. This study paves the way to generate transgenic or gene editing pigs with high efficiency.