RS‐1 enhances CRISPR‐mediated targeted knock‐in in bovine embryos

Targeted knock‐in (KI) can be achieved in embryos by clustered regularly interspaced short palindromic repeats (CRISPR)‐assisted homology directed repair (HDR). However, HDR efficiency is constrained by the competition of nonhomologous end joining. The objective of this study was to explore whether CRISPR‐assisted targeted KI rates can be improved in bovine embryos by exposure to the HDR enhancer RS‐1. In vitro produced zygotes were injected with CRISPR components (300 ng/µl Cas9 messenger RNA and 100 ng/µl single guide RNA against a noncoding region) and a single‐stranded DNA (ssDNA) repair template (100 ng/µl). ssDNA template contained a 6 bp XbaI site insert, allowing targeted KI detection by restriction analysis, flanked by 50 bp homology arms. Following microinjection, zygotes were exposed to 0, 3.75, or 7.5 µM RS‐1 for 24 hr. No differences were noted between groups in terms of development or genome edition rates. However, targeted KI rates were doubled in the group exposed to 7.5 µM RS‐1 compared to the others (52.8% vs. 25% and 23.1%, for 7.5, 0, and 3.75 µM, respectively). In conclusion, transient exposure to 7.5 µM RS‐1 enhances targeted KI rates resulting in approximately half of the embryos containing the intended mutation, hence allowing direct KI generation in embryos.     

Development and characterization of a continuous cell line (EL) from the liver of European eel Anguilla anguilla

In the present study, a new hepatic tissue‐origin cell line from European eel Anguilla anguilla has been developed and characterized. This cell line designated EL has been maintained in Leibovitz L‐15 supplemented with 10% fetal bovine serum over 72 months, and subcultured more than 90 times. The EL cell line consisted predominantly of fibroblast‐like cells, which could survive over 100 days in vitro, and could grow at 15–32°C. The optimum temperature for growth was 27°C. The chromosome analysis revealed a modal diploid karyotype of 2n = 38. The origin of this cell line was confirmed by the 18S recombinant (r)RNA sequencing. The susceptibility test indicated significant cytopathic effects in the EL cells with regard to the Rana grylio virus and the Herpesvirus anguillae. The viral replication was confirmed by transmission electron microscopy and polymerase chain reaction analysis. Following poly (I:C) exposure, the expression levels of the immune‐related molecules interferon regulatory factor‐7 (irf7) and transforming growth factor‐β (TGF‐β) were downregulated in EL cells, whereas the expression levels of the rf3 and the cytochrome P450 (CYP450) were upregulated. All four genes were significantly upregulated following inflammation by lipopolysaccharide (LPS). These data suggested the application of EL cell line for viral identification, as well as for immunodiagnosis and pharmacological targeting.     

基于CRISPR/Cas9的基因分析方法研究进展

自2012年首次证明了CRISPR/Cas9可以在体外进行DNA切割试验以来,CRISPR技术逐渐在基因编辑研究中获得了迅速的发展,除了应用于基因编辑领域之外,它在基因表达调控、基因成像、基因分析等方面也展现出了巨大的应用潜力。尤其在基因分析领域,CRISPR技术由于其精确的基因识别、室温的反应条件、易设计性和操作性等特色,使得一系列新型的基因检测技术得以发展,并取得了超越常规技术的一些检测参数。本文以Cas9蛋白为对象,综述了近些年来在该领域取得的研究进展。主要论述Cas9蛋白的功能、改造、引导RNA(sgRNA)的设计及其在基因分析方法上的应用。     

生菜种子低温处理后耐冻性和脂肪酸合成代谢的关系研究

以生菜(Lactuca sativa)种子为研究对象,通过不同时间的吸水处理分析其含水量变化,再通过程序降温处理,分析不同含水量种子发芽率的差异,以及脂肪酸合成有关基因(FAD2、FAD3、PPT、ELOVL)和冷调节基因ICE1的表达。结果表明,种子含水量随吸水时间增加而升高。程序降温至同样的低温冷冻条件下(-20℃、-22℃),吸水时间小于6 h的种子发芽率较高,而吸水8 h以上的种子发芽率显著降低。种子吸水8 h含水量处于饱和状态,在此状态下种子对低温较为敏感,说明含水量对种子耐冻性有影响。冷冻处理后生菜种子基因表达检测结果表明,脂肪酸去饱和酶基因(FAD2、FAD3)、蛋白质棕榈酰基硫脂酶相关基因(PPT）、长链脂肪酸延伸酶相关基因(ELOVL)的表达水平均随着种子含水量增加呈上升趋势,吸胀10 h的种子表达量最高,此时种子由于高含水量所受冷冻伤害最大。基因ICE1在冷冻处理种子中的表达也随着吸水时间增加而升高,在吸水10 h时种子中表达量到最高水平。综上,种子含水量越高,所受冷冻伤害越大。但种子在低温条件下具有一定的抗冷反应,可通过相关基因的过表达调控合成更多不饱和脂肪酸、抗冻蛋白等提高含水种子耐冻性。     

The microevolutionary response to male‐limited X‐chromosome evolution in Drosophila melanogaster reflects macroevolutionary patterns

Due to its hemizygous inheritance and role in sex determination, the X‐chromosome is expected to play an important role in the evolution of sexual dimorphism and to be enriched for sexually antagonistic genetic variation. By forcing the X‐chromosome to only be expressed in males over >40 generations, we changed the selection pressures on the X to become similar to those experienced by the Y. This releases the X from any constraints arising from selection in females and should lead to specialization for male fitness, which could occur either via direct effects of X‐linked loci or trans‐regulation of autosomal loci by the X. We found evidence of masculinization via up‐regulation of male‐benefit sexually antagonistic genes and down‐regulation of X‐linked female‐benefit genes. Potential artefacts of the experimental evolution protocol are discussed and cannot be wholly discounted, leading to several caveats. Interestingly, we could detect evidence of microevolutionary changes consistent with previously documented macroevolutionary patterns, such as changes in expression consistent with previously established patterns of sexual dimorphism, an increase in the expression of metabolic genes related to mito‐nuclear conflict and evidence that dosage compensation effects can be rapidly altered. These results confirm the importance of the X in the evolution of sexual dimorphism and as a source for sexually antagonistic genetic variation and demonstrate that experimental evolution can be a fruitful method for testing theories of sex chromosome evolution.     

Morphology,Morphogenesis, and Phylogeny of Urosoma caudata (Ehrenberg, 1833) Berger, 1999 (Ciliophora,Hypotrichia) based on a Chinese Population

We report the morphology and morphogenesis of Urosoma caudata (Ehrenberg, 1833) Berger, 1999 based on in vivo observation and protargol impregnation and provide an improved diagnosis of U. caudata based on previous and current work. Urosoma caudata differs from its congeners mainly by the combination of the following features: tail‐like posterior end, colorless cortical granules, and two macronuclear nodules. Urosoma caudata shares most of the ontogenetic features with its congeners: the oral primordium of the opisthe develops apokinetally, and the frontal‐ventral‐transverse cirral anlagen develop in five streaks. However, a unique morphogenetic characteristic is recognizable: the anlagen of three dorsal kineties occur de novo to the left of the parental structures differing from their intrakinetal origin in other Urosoma species. The first record of the 18S rRNA gene sequence for the species is also provided. Phylogenetic analyses based on 18S rRNA gene sequence data suggest that the genus Urosoma is a nonmonophyletic group.     

Melanocortin‐1 receptor (MC1R) genotypes do not correlate with size in two cohorts of medium‐to‐giant congenital melanocytic nevi

Congenital melanocytic nevi (CMN) are cutaneous malformations whose prevalence is inversely correlated with projected adult size. CMN are caused by somatic mutations, but epidemiological studies suggest that germline genetic factors may influence CMN development. In CMN patients from the U.K., genetic variants in MC1R, such as p.V92M and loss‐of‐function variants, have been previously associated with larger CMN. We analyzed the association of MC1R variants with CMN characteristics in two distinct cohorts of medium‐to‐giant CMN patients from Spain (N = 113) and from France, Norway, Canada, and the United States (N = 53), similar at the clinical and phenotypical level except for the number of nevi per patient. We found that the p.V92M or loss‐of‐function MC1R variants either alone or in combination did not correlate with CMN size, in contrast to the U.K. CMN patients. An additional case–control analysis with 259 unaffected Spanish individuals showed a higher frequency of MC1R compound heterozygous or homozygous variant genotypes in Spanish CMN patients compared to the control population (15.9% vs. 9.3%; p = .075). Altogether, this study suggests that MC1R variants are not associated with CMN size in these non‐UK cohorts. Additional studies are required to define the potential role of MC1R as a risk factor in CMN development.