Transgenic pigs expressing ß-xylanase in the parotid gland improve nutrient utilization.

Xylan is one of the main anti-nutritional factors in pig's feed. Although supplementation of ß-xylanase in diet can improve the utilization of nutrients in animals, it is limited by feed cost, manufacturing process and storage stability. To determine whether the expression of endogenous ß-xylanase gene xynB in vivo can improve digestibility of dietary xylan and absorption of nutrients, we produced transgenic pigs which express the xynB from Aspergillus Niger CGMCC1067 in the parotid gland via nuclear transfer. In four live transgenic founders, ß-xylanase activities in the saliva were 0.74, 0.59, 0.37 and 0.24 U/mL, respectively. Compared with non-transgenic pigs, the content of crude protein (CP) in feces reduced by 15.5% (P < 0.05). Furthermore, in 100 of the 271 F1 pigs the xynB gene was detectable. The digestibility of gross energy and CP in F1 transgenic pigs were increased by 5% and 22%, respectively, with the CP content in feces decreased by 6.4%. Taken together, our study showed that the transgenic pigs producing ß-xylanase from parotid gland can reduce the anti-nutritional effect in animal diet and improve the utilization of nutrients.

Protocol for generating three-dimensional induced early ameloblasts using serum-free media and growth factors.

Adult humans cannot regenerate the enamel-forming cell type, ameloblasts. Hence, human induced pluripotent stem cell (hiPSC)-derived ameloblasts are valuable for investigating tooth development and regeneration. Here, we present a protocol for generating three-dimensional induced early ameloblasts (ieAMs) utilizing serum-free media and growth factors. We describe steps for directing hiPSCs toward oral epithelium and then toward ameloblast fate. These cells can form suspended early ameloblast organoids. This approach is critical for understanding, treating, and promoting regeneration in diseases like amelogenesis imperfecta. For complete details on the use and execution of this protocol, please refer to Alghadeer et al.1.

Co-expression of two fibrolytic enzyme genes in CHO cells and transgenic mice.

Cellulose is the main non-starch polysaccharides (NSP) in plant cell walls and acts as anti-nutritional factor in animal feed. However, monogastric animals do not synthesize enzymes that cleave such plant structural polysaccharides and thus waste of resources and pollute the environment. We described the vectors construction and co-expressions of a multi-functional cellulase EGX (with the activities of exo-ß-1,4-glucanase, endo-ß-1,4-glucanase, and endo-ß-1,4-xylanase activities) from mollusca, Ampullaria crossean and a ß-glucosidase BGL1 from Asperjillus niger in CHO cells and the transgenic mice. The recombinant enzymes were synthesised, secreted by the direction of pig PSP signal peptide and functionally active in the eukaryote systems including both of CHO cells and transgenic mice by RT-PCR analysis, western blot analysis and cellulolytic enzymes activities assays. Expressions were salivary glands-specific dependent under the control of pig PSP promoter in transgenic mice. 2A peptide was used as the self-cleaving sequence to mediate co-expression of the fusion genes and the cleavage efficiency was very high both in vitro and in vivo according to the western blot analysis. In summary, we have demonstrated that the single ORF containing EGX and BGL1 were co-expressed by 2A peptide in CHO cells and transgenic mice. It presents a viable technology for efficient disruption of plant cell wall and liberation of nutrients. To our knowledge, this is the first report using 2A sequence to produce multiple cellulases in mammalian cells and transgenic animals.

Single-cell census of human tooth development enables generation of human enamel.

Tooth enamel secreted by ameloblasts (AMs) is the hardest material in the human body, acting as a shield to protect the teeth. However, the enamel is gradually damaged or partially lost in over 90% of adults and cannot be regenerated due to a lack of ameloblasts in erupted teeth. Here, we use single-cell combinatorial indexing RNA sequencing (sci-RNA-seq) to establish a spatiotemporal single-cell census for the developing human tooth and identify regulatory mechanisms controlling the differentiation process of human ameloblasts. We identify key signaling pathways involved between the support cells and ameloblasts during fetal development and recapitulate those findings in human ameloblast in vitro differentiation from induced pluripotent stem cells (iPSCs). We furthermore develop a disease model of amelogenesis imperfecta in a three-dimensional (3D) organoid system and show AM maturation to mineralized structure in vivo. These studies pave the way for future regenerative dentistry.

Role of microparticles in membrane fouling from acidogenesis to methanogenesis phases in an anaerobic baffled reactor.

Microparticles (0.45-10 µm) have been recognized as key foulants in anaerobic membrane bioreactors (AnMBRs). However, their characteristics and fouling behaviors are often understood in single-stage and completely mixed reactors, failing to elucidate the occurrence of microparticles in the multi-stage anaerobic bioprocess. Here, a lab-scale anaerobic baffled reactor with four compartments (C1-C4) was employed to explore the composition and fouling potential of microparticles in different compartments. Photometric analysis showed that the microparticles had an increasing percentage in the total organics of the top supernatant but a decreasing concentration from C1 to C4. Long-term filtration and dead-end filtration tests revealed that the top supernatant in C1 had much higher fouling potential than those in C2-C4. The supernatant microparticles significantly accumulated in the cake layers for each compartment (68-95% of the total organics), particularly the fraction of 1-5 µm, and the fouling rate was positively correlated with the biomass accumulation rate. Based on reactor performance and 16S rRNA gene sequences, a significant bio-phase separation occurred between C1 (acidogenesis) and C2-C4 (methanogenesis). And hydrolytic and fermentative bacteria in the family Veillonellaceae, Streptococcaceae, and Enterobacteriaceae were dominant in the supernatant microparticles, particularly in C1, which had a positive correlation with the fouling rate and biomass accumulation rate. These above results all revealed that the microparticles in the acidogenesis phase had higher fouling potential. In summary, our results suggest that the tactic of pre-hydrolysis and acidification with feedstocks and constructing AnMBRs by coupling with multi-phase anaerobic bioprocesses and membrane units could be beneficial to fouling control.

[Effects of gender and age on protein secretion in saliva of transgenic mice expressing human nerve growth factor].

Nerve growth factor (NGF) can promote the development, differentiation and regeneration of neurons. Recently, in order to efficiently produce human NGF (hNGF) drugs with better efficacy, we created transgenic mice expressing hNGF specifically in their salivary glands, and purified highly active hNGF protein from their saliva. Some studies reported that the NGF secretion in mouse saliva is affected by gender and age. Here, in order to select hNGF transgenic mice with high NGF secretion for saliva collection and hNGF purification, we divided transgenic mice into 4 groups, including 28-day-old young males and females, 63-day-old adult males and females. We compared their saliva volume, total salivary protein amount, salivary mNGF protein amount and salivary hNGF protein amount. The results showed that the saliva volume as well as amounts of total salivary protein, salivary mNGF protein and salivary hNGF protein secreted by 63-day-old transgenic mice were significantly higher than those secreted by sex-match 28-day-old transgenic mice, and the salivary hNGF protein amount secreted by male transgenic mice at the age of 63 days was significantly higher than that of female transgenic mice at the same age; Among 4 groups of mice, 63-day-old male transgenic mice secreted the highest salivary hNGF content, which was about 46 times higher than that secreted by the 28-day-old female transgenic mice. Therefore, 63-day-old male transgenic mice should be selected for saliva collection and hNGF purification.

Production of functional human nerve growth factor from the saliva of transgenic mice by using salivary glands as bioreactors.

The salivary glands of animals have great potential to act as powerful bioreactors to produce human therapeutic proteins. Human nerve growth factor (hNGF) is an important pharmaceutical protein that is clinically effective in the treatment of many human neuronal and non-neuronal diseases. In this study, we generated 18 transgenic (TG) founder mice each carrying a salivary gland specific promoter-driven hNGF transgene. A TG mouse line secreting high levels of hNGF protein in its saliva (1.36 µg/mL) was selected. hNGF protein was successfully purified from the saliva of these TG mice and its identity was verified. The purified hNGF was highly functional as it displayed the ability to induce neuronal differentiation of PC12 cells. Furthermore, it strongly promoted proliferation of TF1 cells, above the levels observed with mouse NGF. Additionally, saliva collected from TG mice and containing unpurified hNGF was able to significantly enhance the growth of TF1 cells. This study not only provides a new and efficient approach for the synthesis of therapeutic hNGF but also supports the concept that salivary gland from TG animals is an efficient system for production of valuable foreign proteins.