Development of genome engineering technologies in cattle: from random to specific

The production of transgenic farm animals(e.g., cattle) via genome engineering for the gain or loss of gene functions is an important undertaking. In the initial stages of genome engineering, DNA micro-injection into one-cell stage embryos(zygotes) followed by embryo transfer into a recipient was performed because of the ease of the procedure.However, as this approach resulted in severe mosaicism and has a low efficiency, it is not typically employed in the cattle as priority, unlike in mice. To overcome the above issue with micro-injection in cattle, somatic cell nuclear transfer(SCNT) was introduced and successfully used to produce cloned livestock. The application of SCNT for the production of transgenic livestock represents a significant advancement, but its development speed is relatively slow because of abnormal reprogramming and low gene targeting efficiency. Recent genome editing technologies(e.g.,ZFN, TALEN, and CRISPR-Cas9) have been rapidly adapted for applications in cattle and great results have been achieved in several fields such as disease models and bioreactors. In the future, genome engineering technologies wil accelerate our understanding of genetic traits in bovine and wil be readily adapted for bio-medical applications in cattle.

Effects of feeding a diet containing Gymnema sylvestre extract: attenuating progression of obesity in C57BL/6J mice

Objective: To investigate the effect of Gymnema sylvestre extract(GS) on initial anti-obesity, liver injury, and glucose homeostasis induced by a high-fat diet(HFD). Methods: The dry powder of GS was extracted with methanol, and gymnemic acid was identified by high performance liquid chromatography as deacyl gymnemic acid. Male C57BL/6J mice that fed on either a normal diet, normal diet containing 1 g/kg GS(CON+GS) HFD, or HFD containing 1.0 g/kg GS(HFD+GS) for 4 weeks were used to test the initial anti-obesity effect of GS. Body weight gain and food intake, and serum levels about lipid and liver injury markers were measured. Histopathology of adipose tissue and liver stained with hematoxylin and eosin(H&E) and oil-red O were analyzed. After 4 weeks of GS extract feeding, intraperitoneal glucose tolerance test(IPGTT) was performed. Results: The methanol extracts of GS exerted significant anti-obesity effects in HFD+GS group. They decreased body weight gain, a lower food and energy efficiency ratio, and showed lower serum levels of total cholesterol(TC), triglyceride(TG), low-density lipoprotein(LDL)-cholesterol, very-low density lipoprotein(VLDL)-cholesterol and leptin compared with the HFD group. The decreases of abdominal as well as epididymal fat weight and adipocyte hypertrophy, lipid droplets in liver, and serum levels of aspartate aminotransferase(AST) and alanine transaminase(ALT) were also observed. The CON+GS group showed an effect of glucose homeostasis compared to the CON group. Conclusions: This study shows that GS provide the possibility as a key role in an initial anti-obesity effects feeding with a HFD.

Combination of cell signaling molecules can facilitate MYOD1-mediated myogenic transdifferentiation of pig fibroblasts

Background:Myogenic transdifferentiation can be accomplished through ectopic MYOD1 expression,which is facilitated by various signaling pathways associated with myogenesis.In this study,we attempted to transdifferentiate pig embryonic fibroblasts(PEFs)myogenically into skeletal muscle through overexpression of the pig MYOD1 gene and modulation of the FGF,TGF-β,WNT,and cAMP signaling pathways.Results:The MYOD1 overexpression vector was constructed based on comparative sequence analysis,demonstrating that pig MYOD1 has evolutionarily conserved domains across various species.Although forced MYOD1 expression through these vectors triggered the expression of endogenous muscle markers,transdifferentiated muscle cells from fibroblasts were not observed.Therefore,various signaling molecules,including FGF2,SB431542,CHIR99021,and forskolin,along with MYOD1 overexpression were applied to enhance the myogenic reprogramming.The modified conditions led to the derivation of myotubes and activation of muscle markers in PEFs,as determined by qPCR and immunostaining.Notably,a sarcomere-like structure was observed,indicating that terminally differentiated skeletal muscle could be obtained from transdifferentiated cells.Conclusions:In summary,we established a protocol for reprogramming MYOD1-overexpressing PEFs into the mature skeletal muscle using signaling molecules.Our myogenic reprogramming can be used as a cell source for muscle disease models in regenerative medicine and the production of cultured meat in cellular agriculture.

Natural Variation in OsPRR37 Regulates Heading Date and Contributes to Rice Cultivation at a Wide Range of Latitudes

Heading date and photoperiod sensitivity are fundamental traits that determine rice adaptation to a wide range of geographic environments. By quantitative trait locus （QTL） mapping and candidate gene analysis using whole- genome re-sequencing, we found that Oryza sativa Pseudo-Response Regulator37 （OsPRR37; hereafter PRR37） is respon- sible for the Early heading7-2 （EH7-2）/Heading date2 （Hd2） QTL which was identified from a cross of late-heading rice ＇Milyang23 （M23）＇ and early-heading rice ＇H143＇. H143 contains a missense mutation of an invariantly conserved amino acid in the CCT （CONSTANS, CO-like, and TOC1） domain of PRR37 protein. In the world rice collection, different types of nonfunctional PRR37 alleles were found in many European and Asian rice cultivars. Notably, the japonica varieties harboring nonfunctional alleles of both Ghd7/Hd4 and PRR37/Hd2 flower extremely early under natural long-day condi- tions, and are adapted to the northernmost regions of rice cultivation, up to 53~ N latitude. Genetic analysis revealed that the effects of PRR37 and Ghd7 alleles on heading date are additive, and PRR37 down-regulates Hd3a expression to suppress flowering under long-day conditions. Our results demonstrate that natural variations in PRR37/Hd2 and GhdT/ Hd4 have contributed to the expansion of rice cultivation to temperate and cooler regions.