Osteoarthritis(OA)is the most common chronic disease,characterized by progressive cartilage breakdown,subchondral bone sclerosis,and aberrant bone outgrowth(Yucesoy et al.,2015;Hussain et al.,2016).OA is one of the le...Osteoarthritis(OA)is the most common chronic disease,characterized by progressive cartilage breakdown,subchondral bone sclerosis,and aberrant bone outgrowth(Yucesoy et al.,2015;Hussain et al.,2016).OA is one of the leading causes of cartilage damage.Patients with severe cartilage damage require transplantation of articular cartilage to improve their quality of life.Type Ⅱ collagen is a major component of articular cartilage and intervertebral discs and plays an important role in the structure and strength of connective tissues that support muscles and joints(Byers,1994).展开更多
Dear Editor,Dogs(Canis familiaris)serve as human companions and are raised to herd livestock,aid hunters,guard homes,perform police and rescue work,and guide the blind.Dogs exhibit close similarities to humans in term...Dear Editor,Dogs(Canis familiaris)serve as human companions and are raised to herd livestock,aid hunters,guard homes,perform police and rescue work,and guide the blind.Dogs exhibit close similarities to humans in terms of metabolic,physiological,and anatomical characteristics,and thus are ideal genetic and clinical models to study human diseases(Tsai et al.,2007).Gene target technology is a powerful tool to create new strains of animals with favorable traits.However,thus far,gene-target dogs have not been developed due to their unique species-specific reproductive characteristics,which limits the applications of dogs especially in the field of biomedical research.Recently,clustered regularly interspaced short palindromic repeats(CRISPRs)/CRISPR-associated(Cas)9 system was applied to edit specific genes with a high efficiency(Cong et al.,2013;Mali et al.,2013).Here we attempt to explore the feasibility of producing gene knockout(KO)dogs by using this technology.Beagle dog,the most widely used breed in biomedical research,was used as our animal model.Myostatin(MSTN)was chosen as the first gene of interest.展开更多
Rapid,precise,and tunable regulation of protein abundance would be significantly useful in a variety of biotechnologies and biomedical applications.Here,we describe a system that allows tunable and rapid drug control ...Rapid,precise,and tunable regulation of protein abundance would be significantly useful in a variety of biotechnologies and biomedical applications.Here,we describe a system that allows tunable and rapid drug control of gene expression for either gene activation or inactivation in mammalian cells.We construct the system by coupling Tet-on 3 G and small molecule-assisted shutoff systems,which can respectively induce transcriptional activation and protein degradation in the presence of corresponding small molecules.This dual-input drug inducer regulation system facilitates a bidirectional control of gene expression.The gene of interest can be precisely controlled by dual small molecules in a broad dynamic range of expression from overexpression to complete silence,allowing gene function study in a comprehensive expression profile.Our results reveal that the bidirectional control system enables sensitive dosage-and time-dependent regulation for either turn-on or shutoff of gene expression.We also apply this system for inducible genome editing and gene activation mediated by clustered regularly interspaced short palindromic repeats.The system provides an integrated platform for studying multiple biological processes by manipulating gene expression in a more flexible way.展开更多
Interspecies chimera through blastocyst complementation could be an alternative approach to create human organs in animals by using human pluripotent stem cells.A mismatch of the major histocompatibility complex of va...Interspecies chimera through blastocyst complementation could be an alternative approach to create human organs in animals by using human pluripotent stem cells.A mismatch of the major histocompatibility complex of vascular endothelial cells between the human and host animal will cause graft rejection in the transplanted organs.Therefore,to achieve a transplantable organ in animals without rejection,creation of vascular endothelial cells derived from humans within the organ is necessary.In this study,to explore whether donor xeno-pluripotent stem cells can compensate for blood vasculature in host animals,we generated rat-mouse chimeras by injection of rat embryonic stem cells(rESCs)into mouse blastocysts with deficiency of Flk-1 protein,which is associated with endothelial and hematopoietic cell development.We found that rESCs could differentiate into vascular endothelial and hematopoietic cells in the rat-mouse chimeras.The whole yolk sac(YS)of Flk-1^EGFP/ECFP rat-mouse chimera was full of rat blood vasculature.Rat genes related to vascular endothelial cells,arteries,and veins,blood vessels formation process,as well as hematopoietic cells,were highly expressed in the YS.Our results suggested that rat vascular endothelial cells could undergo proliferation,migration,and self-assembly to form blood vasculature and that hematopoietic cells could differentiate into B cells,T cells,and myeloid cells in rat-mouse chimeras,which was able to rescue early embryonic lethality caused by Flk-1 deficiency in mouse.展开更多
The prokaryotic clustered regularly interspaced short palindromic repeat(CRISPR)-associated system(Cas)is a simple,robust and efficient technique for gene targeting in model organisms such as zebrafish,mice and rats.I...The prokaryotic clustered regularly interspaced short palindromic repeat(CRISPR)-associated system(Cas)is a simple,robust and efficient technique for gene targeting in model organisms such as zebrafish,mice and rats.In this report,we applied CRISPR technology to rabbits by microinjection of Cas9 mRNA and guided RNA(gRNA)into the cytoplasm of pronuclear-stage embryos.We achieved biallelic gene knockout(KO)rabbits by injection of 1 gene(IL2rg)or 2 gene(IL2rg and RAG1)Cas9 mRNA and gRNA with an efficiency of 100%.We also tested the efficiency of multiple gene KOs in early rabbit embryos and found that the efficiency of simultaneous gene mutation on target sites is as high as 100%for 3 genes(IL2rg,RAG1 and RAG2)and 33.3%for 5 genes(IL2rg,RAG1,RAG2,TIKI1 and ALB).Our results demonstrate that the Cas9/gRNA system is a highly efficient and fast tool not only for single-gene editing but also for multi-gene editing in rabbits.展开更多
基金financially supported by National Key Research and Development Program of China(2017YFA0105103)the Strategic Priority Research Program of the Chinese Academy of Sciences(XDA16030503)+6 种基金Key Research&Development Program of Bioland Laboratory(Guangzhou Regenerative Medicine and Health Guangdong Laboratory)(2018GZR110104004)Science and Technology Planning Project of Guangdong Province,China(2020B1212060052,2017A050501059)Science and Technology Program of Guangzhou,China(202007030003)Research Unit of Generation of Large Animal Disease Models,Chinese Academy of Medical Sciences(2019-I2M-5-025)the Science Foundation for Young Teachers of Wuyi University(2019TD05)Natural Science Foundation of Guangdong Province of China(2019A1515110283)Jiangmen Science and Technology Plan Project(2020JC01030)。
文摘Osteoarthritis(OA)is the most common chronic disease,characterized by progressive cartilage breakdown,subchondral bone sclerosis,and aberrant bone outgrowth(Yucesoy et al.,2015;Hussain et al.,2016).OA is one of the leading causes of cartilage damage.Patients with severe cartilage damage require transplantation of articular cartilage to improve their quality of life.Type Ⅱ collagen is a major component of articular cartilage and intervertebral discs and plays an important role in the structure and strength of connective tissues that support muscles and joints(Byers,1994).
基金supported in part by grants from the National 973 Basic Research Program of China(2011CB944203,2011CB944104)the Ministry of Science and Technology of China(2011ZX09307-304,2011BAI15B02,2013BAK11B02,2012BAI39B01).
文摘Dear Editor,Dogs(Canis familiaris)serve as human companions and are raised to herd livestock,aid hunters,guard homes,perform police and rescue work,and guide the blind.Dogs exhibit close similarities to humans in terms of metabolic,physiological,and anatomical characteristics,and thus are ideal genetic and clinical models to study human diseases(Tsai et al.,2007).Gene target technology is a powerful tool to create new strains of animals with favorable traits.However,thus far,gene-target dogs have not been developed due to their unique species-specific reproductive characteristics,which limits the applications of dogs especially in the field of biomedical research.Recently,clustered regularly interspaced short palindromic repeats(CRISPRs)/CRISPR-associated(Cas)9 system was applied to edit specific genes with a high efficiency(Cong et al.,2013;Mali et al.,2013).Here we attempt to explore the feasibility of producing gene knockout(KO)dogs by using this technology.Beagle dog,the most widely used breed in biomedical research,was used as our animal model.Myostatin(MSTN)was chosen as the first gene of interest.
基金supported by the National Natural Science Foundation of China(81800555,81701580,and 31972926)the National Key R&D Program of China(2018YFC1106400)+1 种基金the Science and Technology Planning Project of Guangdong Province(2015B020229002)the Natural Science Foundation of Guangdong Province(2014A030312013 and 2018A030313128)
文摘Rapid,precise,and tunable regulation of protein abundance would be significantly useful in a variety of biotechnologies and biomedical applications.Here,we describe a system that allows tunable and rapid drug control of gene expression for either gene activation or inactivation in mammalian cells.We construct the system by coupling Tet-on 3 G and small molecule-assisted shutoff systems,which can respectively induce transcriptional activation and protein degradation in the presence of corresponding small molecules.This dual-input drug inducer regulation system facilitates a bidirectional control of gene expression.The gene of interest can be precisely controlled by dual small molecules in a broad dynamic range of expression from overexpression to complete silence,allowing gene function study in a comprehensive expression profile.Our results reveal that the bidirectional control system enables sensitive dosage-and time-dependent regulation for either turn-on or shutoff of gene expression.We also apply this system for inducible genome editing and gene activation mediated by clustered regularly interspaced short palindromic repeats.The system provides an integrated platform for studying multiple biological processes by manipulating gene expression in a more flexible way.
基金financially supported by the Strategic Priority Research Program of the Chinese Academy of Sciences(XDA16030503)National Key Research and Development Program of China(2017YFA0105103)+5 种基金Key Research&Development Program of Guangzhou Regenerative Medicine and Health Guangdong Laboratory(2018GZR110104004)Science and Technology Planning Project of Guangdong Province,China(2014A030312001,2017B020231001,2017A050501059,2017B030314056)Science and Technology Program of Guangzhou,China(201704030034)Research Unit of Generation of Large Animal Disease Models,Chinese Academy of Medical Sciences(2019-I2M-5-025)the Science and Technology Planning Project of Jiangmen(2017TD02)the Young People Fund of Wuyi University(2019TD05)。
文摘Interspecies chimera through blastocyst complementation could be an alternative approach to create human organs in animals by using human pluripotent stem cells.A mismatch of the major histocompatibility complex of vascular endothelial cells between the human and host animal will cause graft rejection in the transplanted organs.Therefore,to achieve a transplantable organ in animals without rejection,creation of vascular endothelial cells derived from humans within the organ is necessary.In this study,to explore whether donor xeno-pluripotent stem cells can compensate for blood vasculature in host animals,we generated rat-mouse chimeras by injection of rat embryonic stem cells(rESCs)into mouse blastocysts with deficiency of Flk-1 protein,which is associated with endothelial and hematopoietic cell development.We found that rESCs could differentiate into vascular endothelial and hematopoietic cells in the rat-mouse chimeras.The whole yolk sac(YS)of Flk-1^EGFP/ECFP rat-mouse chimera was full of rat blood vasculature.Rat genes related to vascular endothelial cells,arteries,and veins,blood vessels formation process,as well as hematopoietic cells,were highly expressed in the YS.Our results suggested that rat vascular endothelial cells could undergo proliferation,migration,and self-assembly to form blood vasculature and that hematopoietic cells could differentiate into B cells,T cells,and myeloid cells in rat-mouse chimeras,which was able to rescue early embryonic lethality caused by Flk-1 deficiency in mouse.
基金This work was supported by grants from the Ministry of Science and Technology National Basic Research Program of China(973 program)(2011CB944203)National Science and Technology Major Project(2009ZX10004-405)+1 种基金Technology and Information of Guangzhou Municipality(12S134060176,2010U1-E00811-5)Guangzhou Municipality and the Chinese Academy of Sciences(ZNGI-2011-010).
文摘The prokaryotic clustered regularly interspaced short palindromic repeat(CRISPR)-associated system(Cas)is a simple,robust and efficient technique for gene targeting in model organisms such as zebrafish,mice and rats.In this report,we applied CRISPR technology to rabbits by microinjection of Cas9 mRNA and guided RNA(gRNA)into the cytoplasm of pronuclear-stage embryos.We achieved biallelic gene knockout(KO)rabbits by injection of 1 gene(IL2rg)or 2 gene(IL2rg and RAG1)Cas9 mRNA and gRNA with an efficiency of 100%.We also tested the efficiency of multiple gene KOs in early rabbit embryos and found that the efficiency of simultaneous gene mutation on target sites is as high as 100%for 3 genes(IL2rg,RAG1 and RAG2)and 33.3%for 5 genes(IL2rg,RAG1,RAG2,TIKI1 and ALB).Our results demonstrate that the Cas9/gRNA system is a highly efficient and fast tool not only for single-gene editing but also for multi-gene editing in rabbits.
