Gene therapy offers potentially transformative strategies for major human diseases.However,one of the key challenges in gene therapy is developing an effective strategy that could deliver genes into the specific tissu...Gene therapy offers potentially transformative strategies for major human diseases.However,one of the key challenges in gene therapy is developing an effective strategy that could deliver genes into the specific tissue.Here,we report a novel virus-like nanoparticle,the bioorthgonal engineered viruslike recombinant biosome(reBiosome),for efficient gene therapies of cancer and inflammatory diseases.The mutant virus-like biosome(mBiosome)is first prepared by site-specific codon mutation for displaying 4-azido-L-phenylalanine on vesicular stomatitis virus glycoprotein of eBiosome at a rational site,and the reBiosome is then prepared by clicking weak acid-responsive hydrophilic polymer onto the mBiosome via bioorthogonal chemistry.The results show that the reBiosome exhibits reduced virus-like immunogenicity,prolonged blood circulation time and enhanced gene delivery efficiency to weakly acidic foci(like tumor and arthritic tissue).Furthermore,reBiosome demonstrates robust therapeutic efficacy in breast cancer and arthritis by delivering gene editing and silencing systems,respectively.In conclusion,this study develops a universal,safe and efficient platform for gene therapies for cancer and inflammatory diseases.展开更多
Both FRT-FRT and LoxP-LoxP sites that are the target sepuences of site-specific recombinases have been constructed in a vector, called C4LFY, using the recombinant DNA technigue. C4LFY also contains P elements, 2 exon...Both FRT-FRT and LoxP-LoxP sites that are the target sepuences of site-specific recombinases have been constructed in a vector, called C4LFY, using the recombinant DNA technigue. C4LFY also contains P elements, 2 exons and 1 intron of Drosophila yellow gene, yellow promoter and enhancers, and flanking DNA. Since C4LFY made use of two pairs of FRT and LoxP sites, this vector included two site-specific recombination systems. C4LFY was then integrated into Drosophila genome by P-element-mediated germ line transformation. in the presence of the FLP or Cre recombinase, either FLP/FRT or Cre/LoxP recombination reaction was successfully created at the same position in the genome. Using this system, the molecular basis of yellow gene expression and regulation during development have been investigated. Results indicate that the tissue-specific expression of yellow gene is directly regulated by transcriptional enhancers. in addition, the 5’ and 3’ genomic sequences flanking the yellow gene have been展开更多
Recently it has been shown that induction of tissue factor (TF) by TNF in vitro is dependent on a concerted action of NFkB and AP-1. However it remains unknown, if TF can mediate intravascular fibrin formation and if ...Recently it has been shown that induction of tissue factor (TF) by TNF in vitro is dependent on a concerted action of NFkB and AP-1. However it remains unknown, if TF can mediate intravascular fibrin formation and if NFkB and AP-1 are involved in intravascular fibrin formation in vivo. When mice with Meth-A sarcomas were injected with TNF; TF was expressed by vascular endothelium of the tumor,展开更多
To solve the problem of embryonic lethality in conventional gene knockouts, site-specific recombinase （SSR） systems （Cre-loxP, FIp-FRT, and φC31） have been used for tissue-specific gene knockout. With the combina...To solve the problem of embryonic lethality in conventional gene knockouts, site-specific recombinase （SSR） systems （Cre-loxP, FIp-FRT, and φC31） have been used for tissue-specific gene knockout. With the combination of an SSR system and inducible gene expression systems （tetracycline and tamoxifen）, stage-specific knockout and transgenic expression can be achieved. The application of this ＂SSR＋inducible＂ conditional tool to genomic manipulation can be extended in various ways. Alternatives to conditional gene targeting, such as conditional gene trapping, multipurpose conditional alleles, and conditional gene silencing, have been developed. SSR systems can also be used to construct precise disease models with point mutations and chromosomal abnormalities. With these exciting achievements, we are moving towards a new era in which the whole genome can be manipulated as we wish.展开更多
Mutations of the first position T and the third position G in TTGACA, the ' - 35' element of sorghum psbA gene promoter, were induced using chemically synthesized 20 nt oligonucleotide primer. Three mutants we...Mutations of the first position T and the third position G in TTGACA, the ' - 35' element of sorghum psbA gene promoter, were induced using chemically synthesized 20 nt oligonucleotide primer. Three mutants were produced: ATTACA, GTGACA, and ATGACA. Then the protein binding affinity of the mutants and the wild type sorghum psbA gene promoter was tested in a spinach chloroplast protein extract system. Gel retardation assay of the展开更多
The rapid development of crops with multiple transgenic traits arouses the need for an efficient system for creating stacked cultivars. Most major crops rely on classical breeding to introgress the transgene from a la...The rapid development of crops with multiple transgenic traits arouses the need for an efficient system for creating stacked cultivars. Most major crops rely on classical breeding to introgress the transgene from a laboratory variety to the numerous cultivars adapted to different growing regions. Even with vegetative propagated crops, genetic crosses are conducted during varietal improvement prior to vegetative cloning. The probability to assort the ＇x＇ number of transgenic loci into a single genome may seem trivial, （~）x for a diploid species, but given the ＇y＇ number of other nontransgenic traits that breeders also need to assemble into the same genome, the （~）~＊y probability for a ＇breeding stack＇ could quickly make the line conversion process unmanageable. Adding new transgenes onto existing transgenic varieties without creating a new segregating locus would require site-specific integration of new DNA at the existing transgenic locus. Here, we tested a recombinase-mediated gene-stacking scheme in tobacco. Sequential site-specific inte- gration was mediated by the mycobacteriophage Bxbl integrase-catalyzed recombination between attP and attB sites. Transgenic DNA no longer needed after integration was excised by Cre recombinase-mediated recombination of Iox sites. Site-specific integration occurred in -10% of the integration events, with half of those events usable as substrates for a next round of gene stacking. Among the site-specific integrants, however, a third experienced gene silencing. Overall, precise structure and reproducible expression of the sequentially added triple traits were obtained at an overall rate of -3% of the transformed clones--a workable frequency for the development of commercial cultivars. Moreover, since nei- ther the Bxbl-att nor the Cre-lox system is under patent, there is freedom to operate,展开更多
Insect is the largest group of animals on land.Many insect species inflict economical and health losses to humans.Yet many more benefit us by helping to maintain balances in our ecosystem.The benefits that insects off...Insect is the largest group of animals on land.Many insect species inflict economical and health losses to humans.Yet many more benefit us by helping to maintain balances in our ecosystem.The benefits that insects offer remain largely untapped,justifying our continuing efforts to develop tools to better understand their biology and to better manage their activities.Here we focus on reviewing the progresses made in the development of genome engineering tools for model insects.Instead of detailed descriptions of the molecular mechanisms underlying each technical advance,we focus our discussion on the logistics for implementing similar tools in non-model insects.Since none of the tools were developed specific for insects,similar approaches can be applied to other non-model organisms.展开更多
基金supported by the National Natural Science Foundation of China(Grant No.81874303 and No.82173752 W.L.Lu).
文摘Gene therapy offers potentially transformative strategies for major human diseases.However,one of the key challenges in gene therapy is developing an effective strategy that could deliver genes into the specific tissue.Here,we report a novel virus-like nanoparticle,the bioorthgonal engineered viruslike recombinant biosome(reBiosome),for efficient gene therapies of cancer and inflammatory diseases.The mutant virus-like biosome(mBiosome)is first prepared by site-specific codon mutation for displaying 4-azido-L-phenylalanine on vesicular stomatitis virus glycoprotein of eBiosome at a rational site,and the reBiosome is then prepared by clicking weak acid-responsive hydrophilic polymer onto the mBiosome via bioorthogonal chemistry.The results show that the reBiosome exhibits reduced virus-like immunogenicity,prolonged blood circulation time and enhanced gene delivery efficiency to weakly acidic foci(like tumor and arthritic tissue).Furthermore,reBiosome demonstrates robust therapeutic efficacy in breast cancer and arthritis by delivering gene editing and silencing systems,respectively.In conclusion,this study develops a universal,safe and efficient platform for gene therapies for cancer and inflammatory diseases.
文摘Both FRT-FRT and LoxP-LoxP sites that are the target sepuences of site-specific recombinases have been constructed in a vector, called C4LFY, using the recombinant DNA technigue. C4LFY also contains P elements, 2 exons and 1 intron of Drosophila yellow gene, yellow promoter and enhancers, and flanking DNA. Since C4LFY made use of two pairs of FRT and LoxP sites, this vector included two site-specific recombination systems. C4LFY was then integrated into Drosophila genome by P-element-mediated germ line transformation. in the presence of the FLP or Cre recombinase, either FLP/FRT or Cre/LoxP recombination reaction was successfully created at the same position in the genome. Using this system, the molecular basis of yellow gene expression and regulation during development have been investigated. Results indicate that the tissue-specific expression of yellow gene is directly regulated by transcriptional enhancers. in addition, the 5’ and 3’ genomic sequences flanking the yellow gene have been
文摘Recently it has been shown that induction of tissue factor (TF) by TNF in vitro is dependent on a concerted action of NFkB and AP-1. However it remains unknown, if TF can mediate intravascular fibrin formation and if NFkB and AP-1 are involved in intravascular fibrin formation in vivo. When mice with Meth-A sarcomas were injected with TNF; TF was expressed by vascular endothelium of the tumor,
基金Project supported by the National Natural Science Foundation of China(Nos.30871436,30973297,and 31171194)the National Basic Research Program(973)of China(No.2010CB945002)+1 种基金the Ministry of Education of China(No.200804220011)Shandong Provincial Science and Technology Key Program(No.2009GG10003039),China
文摘To solve the problem of embryonic lethality in conventional gene knockouts, site-specific recombinase （SSR） systems （Cre-loxP, FIp-FRT, and φC31） have been used for tissue-specific gene knockout. With the combination of an SSR system and inducible gene expression systems （tetracycline and tamoxifen）, stage-specific knockout and transgenic expression can be achieved. The application of this ＂SSR＋inducible＂ conditional tool to genomic manipulation can be extended in various ways. Alternatives to conditional gene targeting, such as conditional gene trapping, multipurpose conditional alleles, and conditional gene silencing, have been developed. SSR systems can also be used to construct precise disease models with point mutations and chromosomal abnormalities. With these exciting achievements, we are moving towards a new era in which the whole genome can be manipulated as we wish.
文摘Mutations of the first position T and the third position G in TTGACA, the ' - 35' element of sorghum psbA gene promoter, were induced using chemically synthesized 20 nt oligonucleotide primer. Three mutants were produced: ATTACA, GTGACA, and ATGACA. Then the protein binding affinity of the mutants and the wild type sorghum psbA gene promoter was tested in a spinach chloroplast protein extract system. Gel retardation assay of the
文摘The rapid development of crops with multiple transgenic traits arouses the need for an efficient system for creating stacked cultivars. Most major crops rely on classical breeding to introgress the transgene from a laboratory variety to the numerous cultivars adapted to different growing regions. Even with vegetative propagated crops, genetic crosses are conducted during varietal improvement prior to vegetative cloning. The probability to assort the ＇x＇ number of transgenic loci into a single genome may seem trivial, （~）x for a diploid species, but given the ＇y＇ number of other nontransgenic traits that breeders also need to assemble into the same genome, the （~）~＊y probability for a ＇breeding stack＇ could quickly make the line conversion process unmanageable. Adding new transgenes onto existing transgenic varieties without creating a new segregating locus would require site-specific integration of new DNA at the existing transgenic locus. Here, we tested a recombinase-mediated gene-stacking scheme in tobacco. Sequential site-specific inte- gration was mediated by the mycobacteriophage Bxbl integrase-catalyzed recombination between attP and attB sites. Transgenic DNA no longer needed after integration was excised by Cre recombinase-mediated recombination of Iox sites. Site-specific integration occurred in -10% of the integration events, with half of those events usable as substrates for a next round of gene stacking. Among the site-specific integrants, however, a third experienced gene silencing. Overall, precise structure and reproducible expression of the sequentially added triple traits were obtained at an overall rate of -3% of the transformed clones--a workable frequency for the development of commercial cultivars. Moreover, since nei- ther the Bxbl-att nor the Cre-lox system is under patent, there is freedom to operate,
基金supported by a grant from the National Natural Science Foundation of China(No.NSFC #31371364)
文摘Insect is the largest group of animals on land.Many insect species inflict economical and health losses to humans.Yet many more benefit us by helping to maintain balances in our ecosystem.The benefits that insects offer remain largely untapped,justifying our continuing efforts to develop tools to better understand their biology and to better manage their activities.Here we focus on reviewing the progresses made in the development of genome engineering tools for model insects.Instead of detailed descriptions of the molecular mechanisms underlying each technical advance,we focus our discussion on the logistics for implementing similar tools in non-model insects.Since none of the tools were developed specific for insects,similar approaches can be applied to other non-model organisms.
