ABSTRACT
Genome-editing technologies have revolutionized the biomedical sciences by providing researchers with the ability to quickly and efficiently modify genes. While programmable nucleases can be introduced into cells using a variety of techniques, their delivery as purified proteins is an effective approach for limiting off-target effects. Here, we describe step-by-step procedures for manufacturing and delivering genome-modifying proteins-including Cas9 ribonucleoproteins (RNPs) and TALE and zinc-finger nucleases-into mammalian cells. Protocols for combining Cas9 RNP with naturally recombinogenic adeno-associated virus (AAV) donor vectors for the seamless insertion of transgenes by homology-directed genome editing are also provided.
Subject(s)
CD4-Positive T-Lymphocytes/metabolism , CRISPR-Associated Protein 9/genetics , Gene Editing/methods , Gene Transfer Techniques , Genetic Vectors/administration & dosage , Transgenes , Zinc Finger Nucleases/genetics , CD4-Positive T-Lymphocytes/cytology , CRISPR-Associated Protein 9/administration & dosage , Cell Differentiation , Cells, Cultured , Genome, Human , Humans , Transcription Activator-Like Effector Nucleases/genetics , Zinc Finger Nucleases/administration & dosageABSTRACT
Zika virus (ZIKV) is associated with neonatal microcephaly and Guillain-Barré syndrome1,2. While progress has been made in understanding the causal link between ZIKV infection and microcephaly3-9, the life cycle and pathogenesis of ZIKV are less well understood. In particular, there are conflicting reports on the role of AXL, a TAM family kinase receptor that was initially described as the entry receptor for ZIKV10-22. Here, we show that while genetic ablation of AXL protected primary human astrocytes and astrocytoma cell lines from ZIKV infection, AXL knockout did not block the entry of ZIKV. We found, instead, that the presence of AXL attenuated the ZIKV-induced activation of type I interferon (IFN) signalling genes, including several type I IFNs and IFN-stimulating genes. Knocking out type I IFN receptor α chain (IFNAR1) restored the vulnerability of AXL knockout astrocytes to ZIKV infection. Further experiments suggested that AXL regulates the expression of SOCS1, a known type I IFN signalling suppressor, in a STAT1/STAT2-dependent manner. Collectively, our results demonstrate that AXL is unlikely to function as an entry receptor for ZIKV and may instead promote ZIKV infection in human astrocytes by antagonizing type I IFN signalling.
Subject(s)
Astrocytes/virology , Interferon Type I/immunology , Proto-Oncogene Proteins/immunology , Receptor Protein-Tyrosine Kinases/immunology , Signal Transduction , Zika Virus/pathogenicity , Astrocytes/immunology , Cell Line , Cells, Cultured , Gene Expression Regulation , Gene Knockout Techniques , Humans , Proto-Oncogene Proteins/genetics , Receptor Protein-Tyrosine Kinases/genetics , Receptor, Interferon alpha-beta/genetics , STAT1 Transcription Factor/genetics , STAT1 Transcription Factor/metabolism , Suppressor of Cytokine Signaling 1 Protein/genetics , Suppressor of Cytokine Signaling 1 Protein/metabolism , Axl Receptor Tyrosine KinaseABSTRACT
OBJECTIVE: To study the effect of the ethanol extract of stir-bake to yellowish Meliae Toosendan Fructus on nerve system and its mechanism. METHODS: The effect of the ethanol extract on sensory nerve was carried out through ache models induced by hot board method and radiant heat stimulation method in mice. The thermalgesia liminal value was investigated. The effect of the ethanol extract on the A-delta fiber and C fiber was measured by electrical stimulation procedure. Motor nerve conduction velocity (NCV) was measured by indirect detection method in vivo. The pathology changes of the motor nerve were observed by transmission electron microscope and the silver stain test. RESULTS: The ethanol extract of Meliae Toosendan Fructus could increase the thermalgesia liminal value of mice and reduce the conduction velocity of motor nerves. Meanwhile, pathology results showed the changes of the fiber of motor nerve, including demyelination and the number of Schwann cells dropping. CONCLUSION: The ethanol extract of stir-bake to yellowish Meliae Toosendan Fructus can reduce the pain sensitivity of mice and slow down NCV, which may be related to decreasing of the number of Schwann cells.
Subject(s)
Meliaceae/chemistry , Neural Conduction/drug effects , Pain Threshold/drug effects , Pain/physiopathology , Plant Extracts/pharmacology , Sciatic Nerve/physiopathology , Animals , Cell Count , Female , Fruit/chemistry , Male , Mice , Motor Neurons/physiology , Nerve Fibers, Unmyelinated/physiology , Neural Conduction/physiology , Pain Measurement , Plant Extracts/administration & dosage , Plant Extracts/isolation & purification , Rats , Rats, Sprague-Dawley , Schwann Cells/physiology , Sciatic Nerve/drug effectsABSTRACT
OBJECTIVE: To observe the trends in blood pressure (BP) and prevalence of hypertension among Chinese children and adolescents. METHODS: Data were extracted from the China Health and Nutrition Survey conducted from 1991 to 2004; 8247 children and adolescents aged 6-17 years were selected for this study. Multivariate linear regression analysis and multivariate logistic regression analysis were performed to evaluate the secular trends in BP levels and prevalence of hypertension, respectively. RESULTS: During the study period, there was an upward trend in BP in Chinese children and adolescents. After adjustment for gender, age and weight status, the prevalence of pre-hypertension and hypertension increased dramatically from 1991 to 2004, with average relative increases of 6.38% and 8.13% in children and adolescents, respectively. Overweight was strongly associated with pre-hypertension and hypertension in comparison with normal weight, with odds ratios (95% confidence intervals) of 2.21 (1.58-3.11) and 4.13 (3.32-5.13), respectively. CONCLUSION: BP levels and prevalence of hypertension increased dramatically among Chinese children and adolescents from 1991 to 2004.
