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Generation of CRISPR/Cas9-mediated lactoferrin-targeted mice by pronuclear injection of plasmid pX330

Mengxu GE,Fei LIU,Fei CHANG,Zhaolin SUN,Jing FEI,Ying GUO,Yunping DAI,Zhengquan YU,Yaofeng ZHAO,Ning LI,Qingyong MENG

《农业科学与工程前沿（英文）》 2015年 第2卷 第3期   页码 242-248 doi: 10.15302/J-FASE-2015059

摘要： Lactoferrin is a member of the transferrin family of multifunctional iron binding glycoproteins. While numerous physiological functions have been described for lactoferrin, the mechanisms underlying these functions are not clear. To further study the functions and mechanisms of lactoferrin, we modified the lactoferrin promoter of mice using the CRISPR/Cas9 system to reduce or eliminate lactoferrin expression. Seven mice with lactoferrin promoter mutations were obtained with an efficiency of 24% (7/29) by injecting the plasmid pX330, expressing a small guide RNA and human codon-optimized SpCas9, into fertilized eggs of mice. Plasmid integration and off-targeting of pX330 were not detected. These results confirmed that pronuclear injection of a circular plasmid is a feasible and efficient method for targeted mutagenesis in mice.

关键词： lactoferrin     promoter     CRISPR/Cas9     plasmid pX330    

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Construction of Hsp90β gene specific silencing plasmid and its transfection efficiency

JI Yewei, NIE Bin, LI Ping, ZHOU Yuanguo, XU Xiaoyu

《医学前沿（英文）》 2007年 第1卷 第3期   页码 253-257 doi: 10.1007/s11684-007-0048-9

摘要： The purpose of this work was to construct the plasmid that could direct the synthesis of siRNA-like transcripts and thus mediate strong and specific repression of human heat shock protein 90β (Hsp90β) gene expression and to compare the transfection efficiency of the plasmids in varying conditions of transfection. Three 64 nt oligos corresponding to different regions of the target gene were chemically synthesized and annealed and were then ligated with pSUPER EGFP1 plasmid and double-digested with HindIII and Π. Recombinant plasmids were transformed into , DH5a, and the colonies were picked and grown in the Amp-agarose. The presence of positive clones was checked by the means of endodigestion and sequencing. Three cell strains, HepG2, Human umbilicus vein endothelium cells (HUVEC) and HeK293, were cultured. Then the plasmids were transfected into the cells at different ratios of plasmid to Lipofectamine. The transfection efficiency was measured by detection of enhanced green fluorescence protein (EGFP). The presence of positive recombinant clones were verified by double-digestion and sequencing. The bases inserted into the plasmids were correct and the positive colonies were named pSuper-Hsp90β1, pSuper-Hsp90β2 and pSuper-Hsp90β3. After optimizing the ratio of plasmid to Lipofectamine, we achieved high transfection efficiency in HeK293 cells. Transfection efficiency was still low in the HepG2 cells. In conclusion, the si-RNA-synthesizing plasmids targeting Hsp90β were constructed and transfected into cells with different transfection efficiency.

关键词： HindIII     transfection efficiency     detection     enhanced     different    

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Degradation of extracellular genomic, plasmid DNA and specific antibiotic resistance genes by chlorination

Menglu Zhang, Sheng Chen, Xin Yu, Peter Vikesland, Amy Pruden

《环境科学与工程前沿（英文）》 2019年 第13卷 第3期 doi: 10.1007/s11783-019-1124-5

摘要：

Extracellular DNA structure damaged by chlorination was characterized.

Integrity of extracellular ARG genetic information after chlorination was determined.

Typical chlorine doses will likely effectively diminish extracellular DNA and ARGs.

Plasmid DNA/ARGs were less readily broken down than genomic DNA.

The Bioanalyzer methodology effectively documented damage incurred to DNA.

关键词： Antibiotic resistance     Antibiotic resistance genes (ARGs)     Extracellular DNA/ARGs     Chlorination    

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Influences and mechanisms of nanofullerene on the horizontal transfer of plasmid-encoded antibiotic resistance

Qingkun Ji, Caihong Zhang, Dan Li

《环境科学与工程前沿（英文）》 2020年 第14卷 第6期 doi: 10.1007/s11783-020-1287-0

摘要： Abstract • Sub-inhibitory levels of nC60 promote conjugative transfer of ARGs. • nC60 can induce ROS generation, oxidative stress and SOS response. • nC60 can increase cell membrane permeability and alter gene expression. • Results provide evidence of nC60 promoting antibiotic resistance dissemination. The spread and development of antibiotic resistance globally have led to severe public health problems. It has been shown that some non-antibiotic substances can also promote the diffusion and spread of antibiotic resistance genes (ARGs). Nanofullerene (nC60) is a type of nanomaterial widely used around the world, and some studies have discovered both the biological toxicity and environmental toxicity of nC60. In this study, cellular and molecular biology techniques were employed to investigate the influences of nC60 at sub-minimum inhibitory concentrations (sub-MICs) on the conjugation of ARGs between the E. coli strains. Compared with the control group, nC60 significantly increased the conjugation rates of ARGs by 1.32‒10.82 folds within the concentration range of 7.03‒1800 mg/L. This study further explored the mechanism of this phenomenon, finding that sub-MICs of nC60 could induce the production of reactive oxygen species (ROS), trigger SOS-response and oxidative stress, affect the expression of outer membrane proteins (OMPs) genes, increase membrane permeability, and thus promote the occurrence of conjugation. This research enriches our understanding of the environmental toxicity of nC60, raises our risk awareness toward nC60, and may promote the more rational employment of nC60 materials.

关键词： Nanofullerene     Sub-minimum inhibitory concentrations     Antibiotic resistance genes     Conjugation     Molecular biological techniques    

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Transport of antibiotic resistance plasmids in porous media and the influence of surfactants

Peipei Chen, Chaoqi Chen, Xiqing Li

《环境科学与工程前沿（英文）》 2018年 第12卷 第2期 doi: 10.1007/s11783-017-0986-7

摘要： Transport of engineered antibiotic resistance plasmids in porous media has been reported to potentially cause significant spreading of antibiotic resistance in the environment. In this work, transport of an indigenous resistance plasmid pK5 in porous media was investigated through packed column experiments. At identical ionic strengths in CaCl solutions, the breakthroughs of pK5 from soil columns were very close to those from quartz sand columns, indicating that transport of pK5 in quartz sand and soil was similar. A similarity in transport behavior was also found between pK5 and an engineered plasmid pBR322 that has approximately the same number of base pairs as pK5. The influence of surfactants, a major group of constituents in soil solutions, was examined using an engineered plasmid pcDNA3.1(+)/myc-His A. The impact of an anionic surfactant, sodium dodecyl sulfate (SDS), was negligible at concentrations up to 200 mg·L . Cetyltrimethyl ammonium bromide (CTAB), a cationic surfactant, was found to significantly enhance plasmid adsorption at high concentrations. However, at environmentally relevant concentrations (<1 mg·L ), the effect of this surfactant was also minimal. The negligible impact of surfactants and the similarity between the transport of engineered and indigenous plasmids indicate that under environmentally relevant conditions, indigenous plasmids in soil also have the potential to transport over long distances and lead to the spreading of antibiotic resistance.

关键词： Indigenous plasmid     Transport     Porous media     Surfactants    

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Lentivector-mediated RNAi efficiently downregulates expression of murine cdk4 gene

Feng JIANG PhD , Xuezhen WANG PhD , Zheng XUE MD , Suming ZHANG PhD , Siyu FANG BM , Min ZHANG MD, PhD ,

《医学前沿（英文）》 2009年 第3卷 第3期   页码 287-291 doi: 10.1007/s11684-009-0050-5

摘要： In order to explore the role of cyclin-dependent kinase 4 (cdk4) in neurodegenerative diseases, lentiviral-delivered RNA interference (RNAi) was used to silence the expression of the murine cdk4 gene . Three cdk4-shRNAs of mouse and a negative sequence were designed. After synthesis and annealing, double strand oligonucleotides were cloned into a linearized pSIH1-H1-copGFP shRNA vector. It was confirmed by polymerase chain reaction (PCR) and sequencing that three pairs of cdk4-shRNAs and a negative shRNA were correctly inserted into the pSIH1-H1-copGFP vector. The above recombinants were transfected by lipofectamine into BV-2 cells. The gene silencing efficacy rates of the 3 targets were compared by Western blotting. The cdk4-siRNA2 was the most effective in silencing cdk4. The optimized pSIH1-cdk4-siRNA2 and pSIH-negative-siRNA were co-transfected into 293T cells with the lentiviral packaging plasmids respectively. The culture supernatant was harvested and condensed at the 24th and 48thh after transfection. Interference efficiency of the lentivirus expressing cdk4-siRNA was determined by reverse transcriptase-PCR (RT-PCR) and Western blotting in BV-2 cells. Lentivector-mediated RNAi could efficiently down-regulate the expression of the murine cdk4 gene , which provides a potential tool for studying and treating cdk4-related diseases.

关键词： cyclin-dependent kinase 4     RNA interference     plasmid     lentiviral vector    

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标题 作者 时间 类型 操作

Generation of CRISPR/Cas9-mediated lactoferrin-targeted mice by pronuclear injection of plasmid pX330

Mengxu GE,Fei LIU,Fei CHANG,Zhaolin SUN,Jing FEI,Ying GUO,Yunping DAI,Zhengquan YU,Yaofeng ZHAO,Ning LI,Qingyong MENG

期刊论文

Construction of Hsp90β gene specific silencing plasmid and its transfection efficiency

JI Yewei, NIE Bin, LI Ping, ZHOU Yuanguo, XU Xiaoyu

期刊论文

Degradation of extracellular genomic, plasmid DNA and specific antibiotic resistance genes by chlorination

Menglu Zhang, Sheng Chen, Xin Yu, Peter Vikesland, Amy Pruden

期刊论文

Influences and mechanisms of nanofullerene on the horizontal transfer of plasmid-encoded antibiotic resistance

Qingkun Ji, Caihong Zhang, Dan Li

期刊论文

Transport of antibiotic resistance plasmids in porous media and the influence of surfactants

Peipei Chen, Chaoqi Chen, Xiqing Li

期刊论文

Lentivector-mediated RNAi efficiently downregulates expression of murine cdk4 gene

Feng JIANG PhD , Xuezhen WANG PhD , Zheng XUE MD , Suming ZHANG PhD , Siyu FANG BM , Min ZHANG MD, PhD ,

期刊论文