Phaeocystis globosa Scherffel is one of the common harmful algae species in coastal waters of the southeastern China.In this study,sandwich hybridization integrated with nuclease protection assay(NPA-SH)was used to qu...Phaeocystis globosa Scherffel is one of the common harmful algae species in coastal waters of the southeastern China.In this study,sandwich hybridization integrated with nuclease protection assay(NPA-SH)was used to qualitatively and quantitatively detect P. globosa.Results showed that this method had good applicability and validity in analyzing the samples from laboratory cultures and from fields.The linear regression equation for P.globosa was obtained,and the lowest detection number of cells was 1.8×104 c...展开更多
The transcription activator-like effector nuclease （TALEN） technique combined with the somatic cel nuclear transfer （SCNT） method has been successfuly applied for creating geneticaly modiifed pigs. However, method...The transcription activator-like effector nuclease （TALEN） technique combined with the somatic cel nuclear transfer （SCNT） method has been successfuly applied for creating geneticaly modiifed pigs. However, methods for isolating cels with bialelic indels requires further improvement because of the relatively low enrichment efifciency of mutated somatic cels. Moreover, little is known regarding the off-target effects of the TALEN system and the heredity of TALEN-modiifed pigs. In this study, an efifcient method to increase the enrichment efifciency of TALEN-mediated bialelic knockout （KO） cels was established, and corresponding geneticaly modiifed pigs with the expected genotype were generated whose off-target effect, fertility and heredity characteristics were aslo evaluated. Two TALEN pairs were constructed to target the porcine α-1,3-galactosyltransferase （GGTA1） gene locus. TALEN mRNA was transfected into the ear ifbroblasts folowed by the enrichment of α-Gal nul cels of minipigs using isolectin B4 （IB4） lectin and magnetic beads. A total of 115 cel colonies were formed and validated to beGGTA1 KO cels by sequencing and 10 bialelic KO cel colonies were used as nuclear donors for SCNT. ThirtyGGTA1 bialelic KO piglets were successfuly delivered and grew normaly. Seventeen potential off-target sites were investigated, and no off-target events were detected in the live piglets. To determine the fertility and heredity characteristics of TALEN-modiifed pigs, 10 mature founders were mated with each other and the mutations were determined to be transmitted to the F1 piglets. We established a robust and safe technology for developing geneticaly modiifed pig lines with expected genotypes for agricultural breeding and biomedical application.展开更多
Plant senescence- or PCD-associated nucleases share significant homology with nucleases from different organisms. However, knowledge of their function is limited. Intracellular localization of the Arabidopsis senescen...Plant senescence- or PCD-associated nucleases share significant homology with nucleases from different organisms. However, knowledge of their function is limited. Intracellular localization of the Arabidopsis senescence- and PCD-associated nuclease BFN1 was investigated. Analysis of BFN1-GFP localization in transiently transformed tobacco protoplasts revealed initial localization in filamentous structures spread throughout the cytoplasm, which then clustered around the nuclei as the protoplasts senesced. These filamentous structures were identified as being of ER origin. In BFN1- GFP-transgenicArabidopsis plants, similar localization of BFN1-GFP was observed in young leaves, that is, in filamentous structures that reorganized around the nuclei only in senescing cells. In late senescence, BFN1-GFP was localized with fragmented nuclei in membrane-wrapped vesicles. BFNI＇s postulated function as a nucleic acid-degrading enzyme in senescence and PCD is supported by its localization pattern. Our results suggest the existence of a dedicated compartment mediating nucleic acid degradation in senescence and PCD processes.展开更多
Large scale genomic aberrations including duplication,deletion,translocation,and other structural changes are the cause of a subtype of hereditary genetic disorders and contribute to onset or progress of cancer.The cu...Large scale genomic aberrations including duplication,deletion,translocation,and other structural changes are the cause of a subtype of hereditary genetic disorders and contribute to onset or progress of cancer.The current prime editor,PE2,consisting of Cas9-nickase and reverse transcriptase enables efficient editing of genomic deletion and insertion,however,at small scale.Here,we designed a novel prime editor by fusing reverse transcriptase(RT)to nuclease wild-type Cas9(WT-PE)to edit large genomic fragment.展开更多
Nuclease effects on the cell internalization of single-walled carbon nanotubes(SWNTs)functionalized with fluorescent-labeled DNA in serum containing cell growth media were examined.When Cy3-labeled DNA-functionalized ...Nuclease effects on the cell internalization of single-walled carbon nanotubes(SWNTs)functionalized with fluorescent-labeled DNA in serum containing cell growth media were examined.When Cy3-labeled DNA-functionalized SWNT conjugates(Cy3DNA-SWNTs)were incubated with HeLa cells in a fatal bovine serum(FBS)medium,a high fl uorescence intensity was obtained from the cells,indicative for the high level inclusion of Cy3DNA-SWNTs.However,the fluorescence intensity was remarkably reduced if Cy3DNA-SWNTs were incubated with cells in the FBS-free medium.Further systematic control experiments revealed that Cy3 dye molecules were released from Cy3DNA-SWNT conjugates by nuclease,and the free Cy3 dyes penetrate into HeLa cell with high efficiency.Although the actual amounts of SWNTs internalized in the cells were almost identical for both cells incubated in the FBS-present and FBS-absent media according to the Raman measurements,one should be cautious to determine the degree of SWNT internalization based on the fl uorescence intensities especially when the coloring dye molecules were linked to oligonucleotides in nuclease containing media.展开更多
Dear Editor,The clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9(CRISPR/Cas9)system,since it was excavated,has been rapidly developed and sparked a revolution in the genome editin...Dear Editor,The clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9(CRISPR/Cas9)system,since it was excavated,has been rapidly developed and sparked a revolution in the genome editing field.In principle,CRISPR/Cas9 system relies on the recognition of specific loci on the genome,which is titled the protospacer adjacent motif(PAM).However,the canonical Streptococcus pyogenes Cas9(SpCas9)nuclease only recognizes NGG or NAG PAMs,rendering an inherent obstacle in amplifying the application of CRISPR/Cas9 technology.展开更多
基金supported by the National Hi-Tech Research and Development Program(863)of China(No.2006AA09Z178,2001AA635090)the National Nat-ural Science Foundation of China(No.40706044)
文摘Phaeocystis globosa Scherffel is one of the common harmful algae species in coastal waters of the southeastern China.In this study,sandwich hybridization integrated with nuclease protection assay(NPA-SH)was used to qualitatively and quantitatively detect P. globosa.Results showed that this method had good applicability and validity in analyzing the samples from laboratory cultures and from fields.The linear regression equation for P.globosa was obtained,and the lowest detection number of cells was 1.8×104 c...
基金supported by the National Basic Research Program of China(973 Program)(2015CB554103 and 2011CBA01004)
文摘The transcription activator-like effector nuclease （TALEN） technique combined with the somatic cel nuclear transfer （SCNT） method has been successfuly applied for creating geneticaly modiifed pigs. However, methods for isolating cels with bialelic indels requires further improvement because of the relatively low enrichment efifciency of mutated somatic cels. Moreover, little is known regarding the off-target effects of the TALEN system and the heredity of TALEN-modiifed pigs. In this study, an efifcient method to increase the enrichment efifciency of TALEN-mediated bialelic knockout （KO） cels was established, and corresponding geneticaly modiifed pigs with the expected genotype were generated whose off-target effect, fertility and heredity characteristics were aslo evaluated. Two TALEN pairs were constructed to target the porcine α-1,3-galactosyltransferase （GGTA1） gene locus. TALEN mRNA was transfected into the ear ifbroblasts folowed by the enrichment of α-Gal nul cels of minipigs using isolectin B4 （IB4） lectin and magnetic beads. A total of 115 cel colonies were formed and validated to beGGTA1 KO cels by sequencing and 10 bialelic KO cel colonies were used as nuclear donors for SCNT. ThirtyGGTA1 bialelic KO piglets were successfuly delivered and grew normaly. Seventeen potential off-target sites were investigated, and no off-target events were detected in the live piglets. To determine the fertility and heredity characteristics of TALEN-modiifed pigs, 10 mature founders were mated with each other and the mutations were determined to be transmitted to the F1 piglets. We established a robust and safe technology for developing geneticaly modiifed pig lines with expected genotypes for agricultural breeding and biomedical application.
文摘Plant senescence- or PCD-associated nucleases share significant homology with nucleases from different organisms. However, knowledge of their function is limited. Intracellular localization of the Arabidopsis senescence- and PCD-associated nuclease BFN1 was investigated. Analysis of BFN1-GFP localization in transiently transformed tobacco protoplasts revealed initial localization in filamentous structures spread throughout the cytoplasm, which then clustered around the nuclei as the protoplasts senesced. These filamentous structures were identified as being of ER origin. In BFN1- GFP-transgenicArabidopsis plants, similar localization of BFN1-GFP was observed in young leaves, that is, in filamentous structures that reorganized around the nuclei only in senescing cells. In late senescence, BFN1-GFP was localized with fragmented nuclei in membrane-wrapped vesicles. BFNI＇s postulated function as a nucleic acid-degrading enzyme in senescence and PCD is supported by its localization pattern. Our results suggest the existence of a dedicated compartment mediating nucleic acid degradation in senescence and PCD processes.
基金We thank Yang Yang for providing python script for HTS data analysis.This work was supported by National Natural Science Foundation of China.No.U19A2002,No.81771220 and No.81974238.
文摘Large scale genomic aberrations including duplication,deletion,translocation,and other structural changes are the cause of a subtype of hereditary genetic disorders and contribute to onset or progress of cancer.The current prime editor,PE2,consisting of Cas9-nickase and reverse transcriptase enables efficient editing of genomic deletion and insertion,however,at small scale.Here,we designed a novel prime editor by fusing reverse transcriptase(RT)to nuclease wild-type Cas9(WT-PE)to edit large genomic fragment.
基金This work was supported by the Nano/Bio Science&Technology Program of MOST(2008-00759)Korean Research Foundation(MOEHRD,KRF-2005-005-J13103 and KRF-2007-313-C00386)KOSEF(2007-8-1158).
文摘Nuclease effects on the cell internalization of single-walled carbon nanotubes(SWNTs)functionalized with fluorescent-labeled DNA in serum containing cell growth media were examined.When Cy3-labeled DNA-functionalized SWNT conjugates(Cy3DNA-SWNTs)were incubated with HeLa cells in a fatal bovine serum(FBS)medium,a high fl uorescence intensity was obtained from the cells,indicative for the high level inclusion of Cy3DNA-SWNTs.However,the fluorescence intensity was remarkably reduced if Cy3DNA-SWNTs were incubated with cells in the FBS-free medium.Further systematic control experiments revealed that Cy3 dye molecules were released from Cy3DNA-SWNT conjugates by nuclease,and the free Cy3 dyes penetrate into HeLa cell with high efficiency.Although the actual amounts of SWNTs internalized in the cells were almost identical for both cells incubated in the FBS-present and FBS-absent media according to the Raman measurements,one should be cautious to determine the degree of SWNT internalization based on the fl uorescence intensities especially when the coloring dye molecules were linked to oligonucleotides in nuclease containing media.
基金supported by the National Transgenic Science and Technology Program(2019ZX08010-003)the Central Publicinterest Scientific Institution Basal Research Fund(Y2020XK17)the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences.
文摘Dear Editor,The clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9(CRISPR/Cas9)system,since it was excavated,has been rapidly developed and sparked a revolution in the genome editing field.In principle,CRISPR/Cas9 system relies on the recognition of specific loci on the genome,which is titled the protospacer adjacent motif(PAM).However,the canonical Streptococcus pyogenes Cas9(SpCas9)nuclease only recognizes NGG or NAG PAMs,rendering an inherent obstacle in amplifying the application of CRISPR/Cas9 technology.
