Production of non-mosaic genome edited porcine embryos by injection of CRISPR/Cas9 into germinal vesicle oocytes

Genetically modified pigs represent a great promise for generating models of human diseases and producing new breeds.Generation of genetically edited pigs using somatic cell nuclear transfer(SCNT)or zygote cytoplasmic microinjection is a tedious process due to the low developmental rate or mosaicism of the founder(FO).Herein,we developed a method termed germinal vesicle oocyte gene editing(GVGE)to produce non-mosaic porcine embryos by editing maternal alleles during the GV to MII transition.Injection of Cas9 mRNA and X-linked Dmd gene-specific gRNA into GV oocytes did not affect their developmental potential.The MII oocytes edited during in vitro maturation(IVM)could develop into blastocysts after parthenogenetic activation(PA)or in vitro fertilization(IVF).Genotyping results indicated that the maternal gene X-linked Dmd could be efficiently edited during oocyte maturation.Up to81.3% of the edited IVF embryos were non-mosaic Dmd gene mutant embryos.In conclusion,GVGE might be a valuable method for the generation of non-mosaic maternal allele edited FO embryos in a short simple step.     

蕾后期和花前期切花菊品种'神马'不同部位叶片光合作用和叶绿素荧光特性的比较

对蕾后期和花前期切花菊(Chrysanthemum morifolium Ramat.)品种'神马'('Jinba')不同部位叶片光合作用参数日变化、叶绿素荧光参数、光响应曲线及参数进行了研究.结果表明:蕾后期和花前期品种'神马'叶片蒸腾速率(Tr)、气孔导度(Gs)和净光合速率(Pn)的日变化均为单峰曲线,峰值出现在10:00或12:00;胞间CO2浓度(Ci)的日变化则先降低后升高,谷值出现在12:00.蕾后期和花前期品种'神马'叶片Tr、Ci和Gs值的平均值总体上随叶片位置降低而逐渐升高;蕾后期不同部位叶片Pn值的平均值差异较小,花前期叶片Pn值的平均值则随叶片位置降低而逐渐降低.随着叶片位置降低,蕾后期和花前期品种'神马'叶片的初始荧光(Fo)、最大荧光(Fm)、可变荧光(Fv)、表观量子效率(AQE)和最大净光合速率(Pmax)以及蕾后期的暗呼吸速率(Rd)均逐渐降低,而花前期的Rd值以及蕾后期和花前期的光补偿点(LCP)均逐渐升高.随着光合有效辐射(PAR)升高,蕾后期不同部位叶片以及花前期中部叶和下部叶的Pn值呈先急剧升高后趋于平稳的变化趋势,而花前期上部叶的Pn值则呈先急剧升高后逐渐下降的变化趋势.研究结果显示:在切花菊设施栽培过程中适当补充光照可提高切花菊品质.     

刺五加及短梗五加根皮有效成分含量对比研究

目的:建立刺五加及短梗五加根皮刺五加苷B、刺五加苷E及异嗪皮啶的高效液相测定方法,测定刺五加及短梗五加中上述成分的含量,对比研究刺五加及短梗五加根中的成分含量差异。方法:采用高效液相色谱法,Agilent TC-C18色谱柱(150 mm×4.6 mm,5μm),以甲醇(A)-水(B)为流动相,梯度洗脱:(0～15 min:20A;15～20 min:21A;20～35 min:30A),流速0.8 mL.min-1,柱温30℃,检测波长210 nm。结果:样品进样量在各自浓度范围内呈良好的线性关系,加样回收率分别为99.1%、97.3%和99.9%,RSD为0.7%和1.3%和0.6%。结论:本法操作简便,分离效果好,可用于刺五加及短梗五加根皮刺五加苷B、刺五加苷E及异嗪皮啶的含量测定。     

脊椎动物的起源与演化研究进展

脊椎动物的起源与演化历来是进化生命科学的核心命题。近年脊椎动物的起源与演化有重大突破。云南澄江寒武纪化石群中的后口类“皇冠西大动物” ,半索动物“云南虫”和“海口虫” ,尾索动物“始祖长江海鞘” ,头索动物“海口华夏鱼”和“中间型中新鱼” ,脊椎动物“凤姣昆明鱼”和“海口鱼” ,论证了普通无脊椎动物向脊椎动物演化过渡的各种中间类型 ,勾勒出一幅较为完整的早期生命演化谱系。西北大学早期生命研究所舒德干教授基于对靠近脊椎动物“源头”时段软躯体后口动物化石系列的研究以及新的发现提出脊椎动物起源分“五步走”的新假说 ,即在脊椎动物的起源的“四步走”前还有更为原始的“一步” :云南澄江出土的古虫动物门化石很可能代表了原口动物和后口动物间的过渡类型。本文即综述了普通无脊椎动物向脊椎动物演化的研究进展。     

口蹄疫病毒前导蛋白的研究进展

FMDV基因组编码的所有蛋白质是以多聚蛋白质的形式产生的。前导蛋白是FMDV基因组编码的第一个具有酶切活性的蛋白质。它是剪切多聚蛋白质的共翻译分子内部的一个蛋白酶。FMDV是在多聚蛋白质的N端剪切前导蛋白。此外,前导蛋白不仅能剪切病毒编码的多聚蛋白,而且能降解宿主细胞中特定的蛋白质,由此极大提高了病毒的毒力。前导蛋白可以抑制I型干扰素的分泌,降低免疫监视系统对FMDV的监视能力,以此逃避宿主的非特异性免疫系统的攻击。关于前导蛋白与细胞凋亡的关系,细胞凋亡产生的eIF4G片段与前导蛋白裂解eIF4G产生的碎片是不同的。     

北京地区首次发现赖型钩端螺旋体

采用分群和分型因子血清经显微镜凝集试验（ＭＡＴ）对由北京市顺义县某农场稻田周围黑线姬鼠肾组织分离获得的４株钩端螺旋体（钩体）进行了血清学分类检定。均属黄疸出血群赖型钩体。这在北京地区尚属首次分离发现。     

Site-specific PEGylation at histidine tags

The efficacy of protein-based medicines can be compromised by their rapid clearance from the blood circulatory system. Achieving optimal pharmacokinetics is a key requirement for the successful development of safe protein-based medicines. Protein PEGylation is a clinically proven strategy to increase the circulation half-life of protein-based medicines. One limitation of PEGylation is that there are few strategies that achieve site-specific conjugation of PEG to the protein. Here, we describe the covalent conjugation of PEG site-specifically to a polyhistidine tag (His-tag) on a protein. His-tag site-specific PEGylation was achieved with a domain antibody (dAb) that had a 6-histidine His-tag on the C-terminus (dAb-His(6)) and interferon α-2a (IFN) that had an 8-histidine His-tag on the N-terminus (His(8)-IFN). The site of PEGylation at the His-tag for both dAb-His(6)-PEG and PEG-His(8)-IFN was confirmed by digestion, chromatographic, and mass-spectral studies. A methionine was also inserted directly after the N-terminal His-tag in IFN to give His(8)Met-IFN. Cyanogen bromide digestion studies of PEG-His(8)Met-IFN were also consistent with PEGylation at the His-tag. By using increased stoichiometries of the PEGylation reagent, it was possible to conjugate two separate PEG molecules to the His-tag of both the dAb and IFN proteins. Stability studies followed by in vitro evaluation confirmed that these PEGylated proteins retained their biological activity. In vivo PK studies showed that all of the His-tag PEGylated samples displayed extended circulation half-lives. Together, our results indicate that site-specific, covalent PEG conjugation at a His-tag can be achieved and biological activity maintained with therapeutically relevant proteins.