Truncated gRNA reduces CRISPR/Cas9-mediated off-target rate for MSTN gene knockout in bovines

The CRISPR/Cas9 mediates efficient gene editing but has off-target effects inconducive to animal breeding. In this study, the efficacy of CRISPR/Cas9 vectors containing different lengths of g RNA in reduction of the off-target phenomenon in the bovine MSTN gene knockout fibroblast cell lines was assessed, providing insight into improved methods for livestock breeding. A 20-bp g RNA was designed for the second exon of the bovine MSTN gene, and CRISPR/Cas9-B was constructed to guide the Cas9 protein to the AGAACCAGGAGAAGATGGACTGG site. The alternative CRISPR/Cas9-19, CRISPR/Cas9-18, CRISPR/Cas9-17 and CRISPR/Cas9-15 vectors were constructed using g RNAs truncated by 1, 2, 3 and 5 bp, respectively. These vectors were then introduced into bovine fetal fibroblasts by the electroporation method, and single cells were obtained by flow cytometry sorting. PCR was performed for each off-target site. All samples were sequenced and analyzed, and finally the efficiency of each vector in target and off-target sites was compared. The CRISPR/Cas9-B vector successfully knocked out the MSTN gene, but the off-target phenomenon was observed. The efficiencies of CRISPR/Cas-B, CRISPR/Cas9-19, CRISPR/Cas9-18, CRISPR/Cas9-17 and CRISPR/Cas9-15 in triggering gene mutations at MSTN targeting sites were 62.16, 17.39, 7.69, 74.29 and 3.85%, respectively;rates of each at the Off-MSTN-1 locus were 52.86, 0, 0, 8.82 and 0%, respectively;all were 0% at the Off-MSTN-2 locus;rates at the Off-MSTN-3 site were 44.87, 51.72, 86.36, 0 and 50%, respectively. The efficiency of the CRISPR/Cas9-17 plasmid in the MSTN site was higher than that in the CRISPR/Cas9-B plasmid, and the effect at the three off-target sites was significantly lower. This study demonstrated that the CRISPR/Cas9-17 plasmid constructed by truncating 3 bp g RNA can effectively reduce the off-target effect without reducing the efficiency of bovine MSTN gene targeting. This finding will provide more effective gene editing strategy for use of CRISPR/Cas9 technology.

A Retrospective Analysis for Different Routes of Administration in Mice-Percutaneous Retro-Orbital, Jugular Catheter, Tail Vein and Femoral Cut Down Injections

Liposomes effectively transport fatty proteins to targeted tissues. Laboratory experiments use multiple methods to administer liposomes, but comparison of these methods is not available. In this retrospective study, we characterized and compared four intravenous administration routes (tail vein, jugular catheter, femoral vein and percutaneous retro-orbital injections) in murine models. ApoE-/- mice were used to compare administration routes. Results indicate that the jugular catheter route delivered the highest amount of liposomes to tissues due to longer period of injections compared to other routes;however, this route failed to remain patent for 8/10 animals. Delivery via tail vein, femoral vein and percutaneous retro-orbital injections resulted in similar accumulation in the organs. When including technical difficulty and expense, percutaneous retro-orbital injections of liposomes are the most convenient and efficacious approach.

CREATE TRANSGENIC RABBITS BY MICROINJECTING HUMAN apoA-Ⅱ GENE INTO FERTILIZED EGGS

Objective To create transgenic rabbits by microinjecting human apolipoprotein A-Ⅱ (apoA-Ⅱ) gene into one-cell embryos, to study apoA-Ⅱ gene function on plasma lipoprotein metabolism and atherosclerosis. Methods Superovulation and synchronization of estrus were induced in female Japanese White Rabbits by injecting hormone, then mating with male. After collected the fertilized eggs, the human apoA-Ⅱ gene was microinjected into the male pronucleus of eggs. The injected eggs were transferred into recipient female rabbits. Last, extract DNA from the new borns ear and determine whether the newborns were transgenic by polymerase chain reaction (PCR) or Southern blot analysis. Results A total of 822 embryos with microinjection of human apoAⅡ gene were implanted into 28 recipient rabbits. The number of surviving newborns was 37. 3 transgenic positive surviving founders were found with human apoA-Ⅱ.

Expression levels of GSTA2 and APOD genes might be associated with carotenoid coloration in golden pheasant(Chrysolophus pictus) plumage

Carotenoids, which generate yellow, orange, and red colors, are crucial pigments in avian plumage. Investigations into genes associated with carotenoid- based coloration in avian species are important; however, such research is difficult because carotenoids cannot be synthetized in vertebrates as they are only derived from dietary sources. Here, the golden pheasant （Chrysolophus pictus） was used as a model in analysis of candidate gene expression profiles implicated in carotenoid binding and deposition. Using mass and Raman spectrometry to confirm the presence of carotenoids in golden pheasant feathers, we found C40H540 and C40H5602 in feathers with yellow to red colors, and in the rachis of iridescent feathers. The global gene expression profiles in golden pheasant skins were analyzed by RNA-seq and all six carotenoid binding candidate genes sequenced were studied by real- time PCR. STAR4, GSTA2, Scarbl, and APOD in feather follicles showed different expressions in red breast and orange nape feathers compared with that of iridescent mantle feathers. Further comparison of golden pheasant yellow rump and Lady Amherst＇s pheasant （Chrysolophus amherstiae） white nape feathers suggested that GSTA2 and APOD played a potential role in carotenoid-based coloration in golden pheasant.

Comparing successful gene knock-in efficiencies of CRISPR/Cas9 with ZFNs and TALENs gene editing systems in bovine and dairy goat fetal fibroblasts

This study aimed to compare the efficiencies of clustered regulatory interspaced short palindromic repeat（CRISPR）/Cas9-mediated gene knock-ins with zinc finger nucleases（ZFNs） and transcription activator-like effector nucleases（TALENs） in bovine and dairy goat fetal fibroblasts. To test the knock-in efficiency, a set of ZFNs and CRISPR/Cas9 plasmids were designed to edit the bovine myostatin（MSTN） gene at exon 2, while a set of TALENs and CRISPR/Cas9 plasmids were designed for editing the dairy goat β-casein gene at exon 2. Donor plasmids utilizing the ZFNs, TALENs, and CRISPR/Cas9 cutting sites were constructed in theGFP-PGK-Neo R plasmid background, including a 5′ and 3′ homologous arm flanking the genes humanized Fat-1（h Fat-1） or enhanced green fluorescent protein（eGFP）. Subsequently, the ZFNs, TALENs, or CRISPR/Cas9 and thehFat-1 or eGFP plasmids were co-transfected by electroporation into bovine and dairy goat fetal fibroblasts. After G418（Geneticin） selection, single cells were obtained by mouth pipetting, flow cytometry or a cell shove. The gene knock-in events were screened by PCR across the homologous arms. The results showed that in bovine fetal fibrobalsts, the efficiencies of ZFNs-mediated eGFP andhFat-1 gene knock-ins were 13.68 and 0%, respectively. The efficiencies of CRISPR/Cas9-mediated eGFP andhFat-1 gene knock-ins were 77.02 and 79.01%, respectively. The eGFP gene knock-in efficiency using CRISPR/Cas9 was about 5.6 times higher than when using the ZFNs gene editing system. Additionally, thehFat-1 gene knock-in was only obtained when using the CRISPR/Cas9 system. The difference of knockin efficiencies between the ZFNs and CRISPR/Cas9 systems were extremely significant（P〈0.01）. In the dairy goat fetal fibroblasts, the efficiencies of TALENs-mediated eGFP andhFat-1 gene knock-ins were 32.35 and 26.47%, respectively. Theefficiencies of eGFP and hFat-1 gene knock-ins using CRISPR/Cas9 were 70.37 and 74.29%, respectively. The knock-in efficiencies difference between the TALENs and CRISPR/Cas9 systems were extremely significant（P〈0.01）. This study demonstrated that CRISPR/Cas9 was more efficient at gene knock-ins in domesticated animal cells than ZFNs and TALENs. The CRISPR/Cas9 technology offers a new era of precise gene editing in domesticated animal cell lines.

Chronic low vitamin intake potentiates cisplatin-induced intestinal epithelial cell apoptosis in WNIN rats

瞄准：为了如果 cisplatin 改变维生素地位并且如果 VR 调制 cisplatin，调查，在 Wistar/NIN (WNIN ) 导致了肠的 apoptosis 和氧化应力雄的老鼠。方法：刚断奶， WNIN 雄的老鼠(n = 12 每组) 为 17 wk 的收到的 adlibitum：与 50% 维生素限制控制饮食(20% 蛋白质) 或一样。他们然后各被细分进二组六只老鼠并且为三 wk 或 PBS (车辆控制) 每周一次管理了 cisplatin (2.61 mg/kg 体重) 。肠的上皮的房间(IEC ) apoptosis 被形态测定法， Annexin-V 绑定， M30 cytodeath 试金和 DNA 破碎监视。绒毛的结构、功能的正直被绒毛高度 / 地窟深度比率和硷性磷酸酯酶的活动估计， lys， ala-dipeptidyl amino-peptidase 分别地。估计 Bcl-2 和 Bax 的改变的 apoptosis，氧化压力参数， caspase-3 活动，和表示的可能的机制是坚定的。结果：Cisplatin 本身减少了血浆维生素层次和他们在与 cisplatin 对待的 VR 动物是最低的。是期望 VR 增加了仅仅绒毛 apoptosis，而 cisplatin 在地窟增加了干细胞 apoptosis。然而， VR 老鼠的 cisplatin 处理在绒毛和地窟区域增加了 apoptosis 并且与 TBARS，蛋白质羰基和 caspase-3 活动的高水平被联系，但是降低 GSH 集中。VR 在 Bcl-2 导致了减少表示被 cisplatin 进一步降低。Bax 表示，由 VR 未受影响在 cisplatin 处理上被增加。粘膜的功能的正直严重地在 cisplatin 被损害对待的 VR 老鼠。结论：维生素的低吸入增加老鼠的敏感到 cisplatin 并且支持肠的上皮的房间 apoptosis。

The protective effects of Xuebijing injection on intestinal injuries of mice exposed to irradiation

Background:Gastrointestinal(GI)injury is one of the most common side effects of radiotherapy.However,there is no ideal therapy method except for symptomatic treatment in the clinic.Xuebijing(XBJ)is a traditional Chinese medicine,used to treat sepsis by injection.In this study,the protective effects of XBJ on radiation-i nduced intestinal injury(RⅢ)and its mechanism were explored.Methods:The effect of XBJ on survival of irradiated C57BL/6 mice was monitored.Histological changes including the number of crypts and the length of villi were evaluated by H&E.The expression of Lgr5^(+)intestinal stem cells(ISCs),Ki67^(+)cells,villin and lysozymes were examined by immunohistochemistry.The expression of cytokines in the intestinal crypt was detected by RT-PCR.DNA damage and apoptosis rates in the small intestine were also evaluated by immunofluorescence.Results:In the present study,XBJ improved the survival rate of the mice after 8.0and 9.0 Gy total body irradiation(TBI).XBJ attenuated structural damage of the small intestine,maintained regenerative ability and promoted proliferation and differentiation of crypt cells,decreased apoptosis rate and reduced DNA damage in the intestine.Elevation of IL-6 and TNF-α was limited,but IL-1,TNF-β and IL-10 levels were increased in XBJ-treated group after irradiation.The expression of Bax and p53 were decreased after XBJ treatment.Conclusions:Taken together,XBJ provides a protective effect on RⅢby inhibiting inflammation and blocking p53-related apoptosis pathway.

Morphine analgesia in male inbred genetic diversity mice recapitulates the among-individual variance in response to morphine in humans

Morphine is a widely used analgesic, but its use in clinical precision medicine is limited by the variance in response among individuals. Although previous studies have shown that individual differences in morphine can be explained in terms of pharmacodynamics and pharmacokinetics, genetic polymorphisms also play an important role. However, the genetic basis of different sensitivity and tolerance susceptibility to morphine remains ambiguous. Using 15 strains of inbred Genetic Diversity(GD) mice,a new resource with wide genetic and phenotypic variation, we demonstrated great variance in sensitivity to morphine analgesia and susceptibility to morphine tolerance between different GD strains. Among-i ndividual variance in response to morphine analgesia in the population can be modeled in GD mice. Two loci respectively may be associated with the among-i ndividual variance in morphine sensitivity and tolerance,confirming the role of genetic factors in among-i ndividual different responses to morphine. These results indicate that GD mice may be a potential tool for the identification of new biomarkers to improve the clinical administration of morphine.

Comparability Assessments of Process Changes Made during Development of Anti-Idiotype Vaccine

Racotumomab monoclonal antibody is a murine anti-idiotypic antibody. This monoclonal antibody mimics N-glycolyl-GM3 gangliosides has been tested in several clinical trials Phase I/II for breast, melanoma and non-small cell lung cancer patients as an anti-idiotypic cancer vaccine. The early production process was performed in vivo from mice ascites fluid. This process was transferred to bioreactor-based method at pilot scale followed to the scale-up of the fermentation. In this work we present a comprehensive molecular characterization of racotumomab MAb produced by the two different production scales in order to determine the impact of the manufacturing process in vaccine performance. We observed differences in glycosylation pattern and charge heterogeneity between racotumomab produced in both scales. Interestingly, these modifications had no significant impact on biological activity elicited in chickens. So, changes in primary structure like glycosylation, charge heterogeneity and oxidation did not affect biological activity of the vaccine.