BackgroundThe CRISPR-Cas9 system is a widely utilized platform for transgenic animal production in various species, although its off-target effects should be addressed. Several applications of this tool have been proposed in model animals but remain insufficient for transgenic livestock production.ResultsHere, we report the first application of single Cas9 nickase (Cas9n) to induce gene insertion at a selected locus in cattle. We identify the main binding sites of a catalytically inactive Cas9 (dCas9) protein in bovine fetal fibroblast cells (BFFs) with chromatin immunoprecipitation sequencing (ChIP-seq). Subsequently, we demonstrate that a single Cas9n-induced single-strand break can stimulate the insertion of the natural resistance-associated macrophage protein-1 (NRAMP1) gene with reduced, but still considerable, off-target effects. Through somatic cell nuclear transfer, we finally obtain transgenic cattle with increased resistance to tuberculosis.ConclusionsOur results contribute to the development of CRISPR-Cas9 system for agriculture applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-1144-4) contains supplementary material, which is available to authorized users.

Follicular atresia is primarily induced by granulosa cell apoptosis, but description of the apoptotic pathway in granulosa cells is incomplete. In this study, we explored the possibility that endoplasmic reticulum (ER) stress could be involved in granulosa cell apoptosis during goat follicular atresia. Immunohistochemical analysis revealed that DNA damage-inducible transcript 3 (DDIT3) and glucose-regulated protein 78 (Grp78) were observed in scattered apoptotic granulosa cells of atretic follicles. Grp78 and DDIT3 mRNA and protein were upregulated in granulosa cells during follicular atresia, although DDIT3 was not significantly different between early atretic and progressed atretic follicles. Spontaneous apoptosis was also observed in vitro in granulosa cells induced by serum deprivation or by the ER stress agent tunicamycin, both inducing similar increases in DDIT3 mRNA. Activating transcription factor-6 (ATF6) and ATF4 mRNAs were significantly increased during granulosa cell apoptosis in vivo; in contrast to ATF6, ATF4 mRNA was attenuated after 16 hr of culture despite the persistence of ER stress. Taken together, ER stress-dependent DDIT3 pathways may play an important role in the regulation of selective granulosa cell apoptosis in goat ovaries during early follicular atresia. Serum deprivation could also increase apoptosis of cultured granulosa cells through the ER stress pathway as both ATF6 and PERK/eIF2α/ATF4 signaling have been implicated in the granulosa cell apoptosis of atretic follicles.

β-Lactoglobulin (BLG) is a major goat’s milk allergen that is absent in human milk. Engineered endonucleases, including transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases, enable targeted genetic modification in livestock. In this study, TALEN-mediated gene knockout followed by gene knock-in were used to generate BLG knockout goats as mammary gland bioreactors for large-scale production of human lactoferrin (hLF). We introduced precise genetic modifications in the goat genome at frequencies of approximately 13.6% and 6.09% for the first and second sequential targeting, respectively, by using targeting vectors that underwent TALEN-induced homologous recombination (HR). Analysis of milk from the cloned goats revealed large-scale hLF expression or/and decreased BLG levels in milk from heterozygous goats as well as the absence of BLG in milk from homozygous goats. Furthermore, the TALEN-mediated targeting events in somatic cells can be transmitted through the germline after SCNT. Our result suggests that gene targeting via TALEN-induced HR may expedite the production of genetically engineered livestock for agriculture and biomedicine.

Background Infertility is a reproductive health problem which affects not only individuals, families and social populations. Recently, the infertility rate in China has a trend of increase year by year, and few studies have reported the infertility rate in Henan Province, China. The aim of this study was to investigate the current prevalence and associated factors of infertility among women of childbearing age in Henan Province, China. Methods This cross-sectional study was conducted from March 2019 to October 2019. We sampled 765 women who were 20–49 years old in eight hospitals of four cities in Henan Province, China. This survey included a questionnaire, physical examination, vaginal ultrasound examinations, and serum anti-Mullerian hormone (AMH) assessment, all of which were conducted under uniform standards by trained personnel. According to the data collected from questionnaire, participants were divided into infertile and fertile groups and analyzed associated factors. Results Among all the 765 participants in this study, the prevalence of infertility was 24.58%. The prevalence of primary infertility was 6.54%, and the prevalence of secondary infertility was 18.04%. In logistic multivariate regression analyses, infertility was associated with age (p < 0.001), history of gynecological surgery (p < 0.001), sweet food (p = 0.003) and decreased ovarian reserve (DOR) (p < 0.001). After further analyses, factors associated with primary infertility were age of marriage (p = 0.006), age of first sexual intercourse (p = 0.003), long-term air-conditioning environment (p < 0.001), decreased ovarian reserve (p = 0.005) and age (p = 0.002). And factors associated with secondary infertility were history of gynecological surgery (p < 0.001), decreased ovarian reserve (p = 0.002), waist-to-hip ratio (WHR) above 0.85 (p = 0.043), delivery times (p = 0.001) and ages (p < 0.001). Conclusion The prevalence of infertility among women aged 20–49 was 24.58% and only 61.17% infertile women sought medical help in Henan Province, China. Age, history of gynecological surgeries and DOR may increase the risk of infertility. Local public health departments and medical professionals need to discharge their duty of reducing the high incidence of infertility and protecting women’s reproductive health.

HMGB1 promoted the proliferation and odontoblastic differentiation of hDPCs.

In this study, the solubilities and densities of a ternary system (NaH2PO4 + CO(NH2)2 + H2O) at 298.15 K was investigated using isothermal solution saturation and moist residues. According to the experimental results, the phase diagram and the diagram of densities versus composition were plotted. It turned out that there were one cosaturated point (CO(NH2)2 + NaH2PO4·2H2O), two univariant curves, and two crystallization regions corresponding to NaH2PO4·2H2O and CO(NH2)2 in this ternary system. The system belonged to a simple eutectic type, in which no double salt was formed. This research filled the blank of phase diagram in this ternary system and demonstrated the absence of double salt in the system.

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Luman/CREB3 recruitment factor (LRF or CREBRF) was identified as a regulator of Luman (or CREB3) that is involved in the unfolded protein response during endoplasmic reticulum stress. Luman is implicated in a multitude of functions ranging from viral infection and immunity to cancer. The biological function of LRF, however, is unknown. In this paper, we report that uteri of pregnant mice and embryos displayed enhanced LRF expression at all stages, and the expressed LRF was found to be localized specifically at implantation sites. On the other hand, uteri of mice induced for delayed implantation or pseudopregnant mice showed low levels of LRF expression, suggesting that LRF mediates uterine receptivity during implantation. Further, expression of LRF was found to be modulated by steroid hormones such as progesterone and estradiol. This study thereby identifies a potential role for LRF in the process of implantation in uteri and development of preimplantation embryos in mice.