Production of transgenic first filial puppies expressing mutated human amyloid precursor protein gene.

Propagation of transgenic animals by germline transmission using assisted reproductive technologies such as in vitro fertilization (IVF) is the most efficient way to produce transgenic colonies for biomedical research. The objective of this study was to generate transgenic puppies from a founder dog expressing the mutated human amyloid precursor protein (mhAPP) gene. Experiment I assessed the characteristics of the semen prepared by freshly diluted, swim-up, and Percoll gradient methods using a computer-assisted semen analyzer (CASA). Motile and progressively motile sperm counts were higher in the Percoll gradient samples (p < 0.05) than in the swim-up and freshly diluted samples. In Experiment II, a total of 59, 70, and 65 presumptive zygotes produced by fresh, Percoll gradient, and swim-up methods, respectively, were transferred to surrogates (5 for each group); the Percoll gradient (27.27%) and swim-up samples (14.29%) showed the highest blastocyst formation rates, while fresh diluted semen did not produce any blastocyst. Experiment III examined the full-term developmental ability of embryos. Among the 5 surrogates in the Percoll gradient group, one (20.0%) became pregnant; it had 4 (6.15%) sacs and delivered 4 (6.15%; 2 males and 2 females) live puppies. Among the 4 puppies, 2 (50.0%) were found to transmit the transgene on their nail and toe under GFP fluorescence. Furthermore, the integration and expression of the mhAPP transgene were examined in the umbilical cords of all the IVF-derived puppies, and the presence of the transgene was only observed in the GFP-positive puppies. Thus, semen prepared by the Percoll method could generate transgenic puppies by male germline transmission using the IVF technique. Our result will help propagate transgenic dogs efficiently, which will foster human biomedical research.

Stochastic anomaly of methylome but persistent SRY hypermethylation in disorder of sex development in canine somatic cell nuclear transfer.

Somatic cell nuclear transfer (SCNT) provides an excellent model for studying epigenomic reprogramming during mammalian development. We mapped the whole genome and whole methylome for potential anomalies of mutations or epimutations in SCNT-generated dogs with XY chromosomal sex but complete gonadal dysgenesis, which is classified as 78, XY disorder of sex development (DSD). Whole genome sequencing revealed no potential genomic variations that could explain the pathogenesis of DSD. However, extensive but stochastic anomalies of genome-wide DNA methylation were discovered in these SCNT DSD dogs. Persistent abnormal hypermethylation of the SRY gene was observed together with its down-regulated mRNA and protein expression. Failure of SRY expression due to hypermethylation was further correlated with silencing of a serial of testis determining genes, including SOX9, SF1, SOX8, AMH and DMRT1 in an early embryonic development stage at E34 in the XY(DSD) gonad, and high activation of the female specific genes, including FOXL2, RSPO1, CYP19A1, WNT4, ERα and ERß, after one postnatal year in the ovotestis. Our results demonstrate that incomplete demethylation on the SRY gene is the driving cause of XY(DSD) in these XY DSD dogs, indicating a central role of epigenetic regulation in sex determination.

Effect of mechanical stimulation on the differentiation of cord stem cells.

In this study, we evaluated the effect of mechanical stimulation on the differentiation of umbilical cord-derived mesenchymal stem cells (UC-MSCs) in osteogenic medium using a Flexcell system that imposed cyclic uniaxial mechanical stimulation at a strain of 0%, 5%, or 10% (5 s of stretch and 15 s of relaxation) for 10 days. The expression of MSC surface antigens (CD73, CD90, and CD105) was significantly decreased as strain increased. Mechanical stimulation inhibited the growth of UC-MSCs and slightly raised lactate dehydrogenase production. Mechanically stimulated groups produced more elastin and sulfated glycosaminoglycan than unstimulated groups and these increases were in proportion to the degree of strain. Reverse transcription-polymerase chain reaction analysis revealed that mechanical stimulation induced a significant increase in the mRNA expression of osteoblast differentiation markers. The mRNA levels of osteopontin, osteonectin, and type I collagen in the 5% and 10% strained groups were significantly higher than those in the 0% strained group. From the Western blot analysis, UC-MSCs produced bone sialoprotein and vimentin in a mechanical strain-dependent manner. Thus, cyclic mechanical loading was able to enhance the differentiation of human UC-MSCs into osteoblast-like cells as determined by osteogenic gene and protein expression. Furthermore, this finding has important implications for the use of the combination of mechanical and osteogenic differentiation media for UC-MSCs in tissue engineering and regenerative medicine.