To knock out β-lactoglobulin (BLG) gene and insert human lactoferrin (hLF) coding sequence at BLG locus of goat, the transcription activator-like effector nucleases (TALEN) mediated recombination was used to edit the BLG gene of goat fetal fibroblast, then as donor cells for somatic cell nuclear transfer. We designed a pair of specific plasmid TALEN-3-L/R for goat BLG exon III recognition sites, and BLC14-TK vector containing a negative selection gene HSV-TK, was used for the knock in of hLF gene. TALENs plasmids were transfected into the goat fetal fibroblast cells, and the cells were screened three days by 2 μg/mL puromycin. DNA cleavage activities of cells were verified by PCR amplification and DNA production sequencing. Then, targeting vector BLC14-TK and plasmids TALEN-3-L/R were co-transfected into goat fetal fibroblasts, both 700 μg/mL G418 and 2 μg/mL GCV were simultaneously used to screen G418-resistant cells. Detections of integration and recombination were implemented to obtain cells with hLF gene site-specific integration. We chose targeting cells as donor cells for somatic cell nuclear transfer. The mutagenicity of TALEN-3-L/R was between 25% and 30%. A total of 335 reconstructed embryos with 6 BLG-/hLF+ targeting cell lines were transferred into 16 recipient goats. There were 9 pregnancies confirmed by ultrasound on day 30 to 35 (pregnancy rate of 39.1%), and one of 50-day-old fetus with BLG-/hLF+ was achieved. These results provide the basis for hLF gene knock-in at BLG locus of goat and cultivating transgenic goat of low allergens and rich hLF in the milk.
Animals ; Animals, Genetically Modified ; genetics ; Female ; Fibroblasts ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Goats ; genetics ; Humans ; Lactoferrin ; genetics ; Lactoglobulins ; genetics ; Milk ; chemistry ; Nuclear Transfer Techniques ; Plasmids ; Pregnancy ; Transfection

Gene knockout by ZFNs (zinc-finger nucleases) is efficient and specific, and successfully applied in more than 10 organisms. Currently, it is unclear whether this technology can be used for knocking-out enhanced green fluorescent protein (EGFP) gene in transgenic goats. Here we constructed and used ZFN-coding plasmids to produce genetic knockouts in the cells of cloned fetus produced from donor cells by microinjection of EGFP gene. Following introduced plasmids into caprine primary cultured fetus fibroblasts by electroporation, targeting of a transgene resulted in sequence mutation. Using the flow cytometric analysis, we confirmed the disappearance of EGFP expression in treated cells. Sequence from PCR products corresponding to targeted site showed that insertion of a G into the exon of EGFP resulted in frame shift mutation. These results suggest that ZFN-mediated gene targeting can apply to caprine fetus fibroblasts, which may open a unique avenue toward the creation of gene knockout goats combining with somatic cell nuclear transfer.
Animals ; Base Sequence ; Cloning, Organism ; Electrophoresis ; Endonucleases ; genetics ; metabolism ; Fetus ; Fibroblasts ; metabolism ; Gene Knockout Techniques ; Gene Targeting ; methods ; Goats ; Green Fluorescent Proteins ; genetics ; Molecular Sequence Data ; Mutation ; Zinc Fingers

Objective To analyze the distribution of class 1,2 and 3 integrons and their gene cassettes,and to explore its relationship with drug resistance in Shigella.Methods Antimicrobial susceptibility was detected by minimal inhibitory concentration (MIC) method.All the genes of integrons and gene cassettes were amplified by polymerase chain reaction (PCR).The amplicons were identified by amplified fragment length polymorphism (AFLP) and sequencing.Results Fifty seven multi-drug resistant strains were identified from a total of 62 Shigella strains (91.9％).Among multi drug resistant strains,52 strains carried integrons of class 1 (91.2 ％) and 55 strains carried integrons of class 2 (96.5％).Only 2 strains carried class 1 integrons alone,5 strains carried class 2 integrons alone and 50 strains had both class 1 and class 2 integrons.Class 3 integrons were not detected.The gene cassettes of typical class 1 integrons,dfrV and dfrA17-aadA5,were detected in 6 strains and 2 strains,respectively.Atypical class 1 integrons with gene cassettes blaOxA30 aadA1 were detected in 44 strains.The typical and atypical class 1 integrons coexisted in 6 Shigella flexneri strains.Gene cassettes for class 2 integrons were dfrA1 sat1-aadA1.Conclusions The multi-drug resistant Shigella strains are widely distributed in Ji'nan,and the atypical class 1 integrons and class 2 integrons are common in these strains.Coexistence of the two integrons is observed in some of the strains.

We compared the efficiency of cloning goat using human lactoferrin (hLF) with genetically modified donor cells marked by single (Neo(r)) or double (Neo(r)/GFP) markers. Single marker expression vector (pBLC14) or dual markers expression vector (pAPLM) was delivered to goat fetal fibroblasts (GFF), and then the transgenic GFF was used as donor cells to produce transgenic goats. Respectively, 58.8% (20/34) and 86.7% (26/30) resistant cell lines confirmed the transgenic integration by PCR. Moreover, pAPLM cells lines were subcultured with several passages, only 20% (6/30) cell lines was observed fluorescence from each cell during the cell passage. Somatic cell nuclear transfer using the donor cells harbouring pBLC14 or pAPLM construct, resulting in a total of 806 reconstructed embryos, a pregnancy rate at 35 d (53.8%, 39.1%) and 60 d (26.9%, 21.7%), and an offspring birth rate (1.9%, 1.4%) with 5 and 7 newborn cloned goats, respectively. Transgene was confirmed by PCR and southern-blot in all cloned offspring. There were no significant differences at the reconstructed embryo fusion rates, pregnancy rates and the birth rate (P > 0.05) between single and double markers groups. The Neo(r)/GFP double markers could improve the reliability for accurately and efficiently selecting the genetically modified donor cells. No adverse effect was observed on the efficiency of transgenic goat production by SCNT using somatic cells transfected with double (Neo(r)/GFP) markers vector.
Animals ; Animals, Genetically Modified ; genetics ; Cloning, Molecular ; Cloning, Organism ; methods ; veterinary ; Fetus ; Fibroblasts ; cytology ; Genetic Markers ; Goats ; embryology ; genetics ; Green Fluorescent Proteins ; genetics ; Humans ; Lactoferrin ; biosynthesis ; genetics ; Neomycin ; Nuclear Transfer Techniques ; veterinary ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection ; veterinary

Objective: To evaluate the effect of Sacubitril/Valsartan for alleviating chronic heart failure(CHF)in elderly patients after acute myocardial infarction(AMI). Methods: A total of 87 elderly patients with AMI-induced CHF treated in Heze Shili Hospital from October 2017 to August 2018 were enrolled and randomly divided into the experimental group(n=42)and the control group(n=45). All patients were given standard AMI treatments, and patients in the experimental group were given Sacubitril/Valsartan(100 mg bid)while those in the control group received Valsartan(80 mg qd). After a follow-up of 12 months, levels of N-terminal pro-brain natriuretic peptide(NT-proBNP), left ventricular end-diastolic diameter(LVDd), left ventricular ejection fraction(LVEF), rates of rehospitalization for heart failure and all-cause mortality were compared between the two groups. Results: Among the 87 patients, 51 patients(58.6%)were male and 36 were female, with an averageage of(67.4±4.0)years.After 12-months of treatment, patients in the experimental group were associated with significantly lower levels of LVDd[(47.86±3.86)mm vs.(50.73±4.39)mm, P<0.05]and NT-proBNP[(793.43±335.43)ng/L vs.(1 068.44±344.46)ng/L, P<0.05]and higher levels of LVEF[(53.74±4.08)% vs.(44.42±7.41)%, P<0.05]than those in the control group.Moreover, the rehospitalization rate for heart failure was markedly higher in the control group than that in the experimental group[15(33.3%)vs.5(11.9%), P<0.05], while the rate of all-cause mortality was similar between the two groups[2(4.8%)vs.3(6.7%), P=0.703]. Conclusions Compared with Valsartan, Sacubitril/Valsartan can reduce the incidence of CHF after AMI, improve left ventricular function, and reduce the rehospitalization rate due to CHF in elderly patients.

In this study, we evaluated the development potential of caprine mammary gland epithelial cells (CMGECs) after transfection and nuclear transfer into enucleated, ovulated oocytes. We first isolated CMGECs from udders of lactating goats which were transfected with expression plasmid for human lacterrin and selected by G418. Then we chose sixteen neomycin resistant lines and induced them with prolactin for the expression of human lactoferrin checked by Western blotting. The donor cells, expressing human lactoferrin of 75 kD, were fused and activated with enucleated ovulated oocytes. Pronuclear-stage reconstructed embryos were transferred into the oviducts of 16 recipient goats. There were fourteen (87.5%), thirteen (81.3%), and ten (62.5%) pregnancies confirmed pregnant by ultrasound on Day 30, 60, and 90, respectively. Three recipients carried the pregnancies to term and delivered one goat each. Nested PCR-RFLP analysis confirmed that all of the kids were clones of the donor cells. These results demonstrated that CMGECs after transfection remain totipotent for nuclear transfer.
Animals ; Cloning, Organism ; methods ; Epithelial Cells ; cytology ; Female ; Goats ; Humans ; Lactoferrin ; biosynthesis ; Mammary Glands, Animal ; cytology ; Nuclear Transfer Techniques ; Pregnancy ; Transfection

Objective To determine the trend of emergency department (ED)mortality of a tertiary general hospital from 2004 to 2014 in order to find the factors that may impact on ED mortality.Methods Mortality in ED was estimated according to the ratio of ED visiting patients to ED deaths.And the data of all ED deaths in 2004,2009 and 2014 were collected.Variance analysis and chi-square test were used for data analysis.Results During the past decade,ED visiting patients was increased significantly by 38.0% in 2014,compared with those in 2004,and the mortality was also increased accordingly from 0.7% in 2004, to 0.9% in 2009,to 1.2% in 2014 (P <0.01).Finally,a total of 1,091 deaths occurred in these three years were included for further evaluation.There were no significant changes in average age and gender distribution,and the average age was 61.9 and the male /female ratio was 1.36∶1 during past decade.The number of adults under 40 years old (18 -39)increased from 7.5% in 2004,to 10.6% in 2009,to 14.4% in 2014 (P <0.05).Both the facilities were upgraded and the number of staffs in ED increased markedly.The cardiovascular illness,cerebrovascular diseases,and sudden death were the leading causes of ED death during past decade.The incidences of trauma and tumor remained unchanged.Average time consumed from onset of illness to arrival to ED didn’t vary significantly during past decade.The study showed no changes in use of ambulance,but remarkable increases in number of non-compliant patients or their family from 18.3% in 2004,to 25.6% in 2009,to 38.3% in 2014 (P <0.01).The percentage of patients in the night time was higher,but there were no significant changes in number of emergency patients in the night time and during holidays in the past decade,but the mean ED stay time increased obviously from 22.4 h in 2004 to 53.3 h in 2014 (P <0.05 ).Conclusions During the past decade,although ED facilities and number of staffs have been improved apparently,ED mortality rate still keeps on escalating. The increase in ED mortality rate may be related to the severely ill patients presenting to ED,the obvious decrease in compliance of patients and the prolonged ED stay time.

Objective:To clarify the clinical efficacy of Yiqi Huoxue recipe in the treatment of liver fibrosis of chronic viral hepatitis.Methods:An open, positive-drug, parallel-controlled study method was applied. A total of 207 cases of liver fibrosis with chronic hepatitis B and C diagnosed with liver biopsy and transient elastography were selected. According to the principle of syndrome differentiation in traditional Chinese medicine, self-made Yiqi Huoxue recipe ( n = 127) and Fuzheng Huayu capsule ( n = 80) were used for the treatment course of 24-48 weeks. Change score of TCM symptom, liver biochemistry, liver stiffness measurement (LSM), and noninvasive liver fibrosis index [aspartate transaminase to platelet ratio index (APRI), and fibrosis-4 score (FIB-4)] were compared between the two groups to evaluate the therapeutic effect of Yiqi Huoxue recipe on liver fibrosis. Results:Yiqi Huoxue recipe group and Fuzheng Huayu capsule group baseline LSM, APRI and FIB-4 was compared, and there was no statistically significant difference between them ( P > 0.05). Yiqi Huoxue recipe and Fuzheng Huayu capsule received patients had improved symptom scores to a certain extent. Hepatic facies, discomfort over liver area, and soreness and weakness of waist and knees ( P < 0.05) was significantly improved in Yiqi Huoxue recipe than Fuzheng Huayu capsule. Liver biochemical indicators (ALT, AST, GGT, ALP) had gradually relapsed with the extension of treatment duration and the normalization rate between the two groups after 24 to 48 weeks had reached 100% vs. 100%, 100% vs. 93.8%, 96.8% vs. 92.3% and 87.5% vs. 81.8%. After 12 weeks of treatment, APRI values ??of both groups had significantly reduced, and after 48 weeks of treatment, LSM values of both groups had significantly improved. Moreover, Yiqi Huoxue recipe FIB-4 score was significantly improved after 48 weeks of treatment, and the difference was statistically significant compared to Fuzheng Huayu capsule group ( P < 0.05). After treatment, LSM, APRI, and FIB-4 total effectiveness in the two groups were 80.0% vs. 63.6%, P = 0.046; 68.4% vs. 52.0%, P = 0.052; 68.4% vs. 62.0%, P = 0.437, respectively. LSM total effectiveness was significantly higher in Yiqi Huoxue recipe treated group than Fuzheng Huayu capsule group. Conclusion:Traditional Chinese medicine Yiqi Huoxue decoction can be used as an optimal treatment for liver fibrosis of chronic viral hepatitis.

Myostatin gene (MSTN) encodes a negative regulator for controlling skeletal muscle growth in animals. In this study, MSTN^-/- homozygous mutants with "double muscle" phenotypic traits and stable inheritance were bred on the basis of MSTN gene editing rabbits, with the aim to establish a method for breeding homozygous progeny from primary MSTN biallelic mutant rabbits. MSTN^-/- primary mutant rabbits were generated by CRISPR/Cas9 gene editing technology. The primary mutant rabbits were mated with wild type rabbits to produce F1 rabbits, whereas the F2 generation homozygous rabbits were bred by half-sibling mating or backcrossing with F1 generation rabbits of the same mutant strain. Sequence analysis of PCR products and its T vector cloning were used to screen homozygous rabbits. The MSTN mutant rabbits with 14-19 week-old were weighed and the difference of gluteus maximus tissue sections and muscle fiber cross-sectional area were calculated and analyzed. Five primary rabbits with MSTN gene mutation were obtained, among which three were used for homozygous breeding. A total of 15 homozygous rabbits (5 types of mutants) were obtained (M2-a: 3; M2-b: 2; M3-a: 2; M7-a: 6; M7-b: 2). The body weight of MSTN^-/- homozygous mutant rabbits aged 14-19 weeks were significantly higher than that of MSTN^+/+ wild-type rabbits of the same age ((2 718±120) g vs. (1 969±53) g, P < 0.01, a 38.0% increase). The mean cross sections of gluteus maximus muscle fiber in homozygous mutant rabbits were not only significantly higher than that of wild type rabbits ((3 512.2±439.2) μm² vs. (1 274.8±327.3) μm², P < 0.01), but also significantly higher than that of MSTN^+/- hemizygous rabbits ((3 512.2±439.2) μm² vs. (2 610.4±604.4) μm², P < 0.05). In summary, five homozygous mutants rabbits of MSTN^-/- gene were successfully bred, which showed a clear lean phenotype. The results showed that the primary breeds were non-chimeric mutant rabbits, and the mutant traits could be inherited from the offspring. MSTN^-/- homozygous mutant rabbits of F2 generation could be obtained from F1 hemizygous rabbits by inbreeding or backcrossing. The progenies of the primary biallelic mutant rabbits were separated into two single-allelic mutants, both of which showed a "double-muscle" phenotype. Thus, this study has made progress in breeding high-quality livestock breeds with gene editing technology.
Animals ; CRISPR-Cas Systems/genetics* ; Gene Editing ; Muscle, Skeletal/metabolism* ; Mutation ; Myostatin/metabolism* ; Phenotype ; Rabbits