Most viruses and transposons serve as effective carriers for the introduction of foreign DNA up to 11 kb into vertebrate genomes.However,their activity markedly diminishes with payloads exceeding 11 kb.Expanding the p...Most viruses and transposons serve as effective carriers for the introduction of foreign DNA up to 11 kb into vertebrate genomes.However,their activity markedly diminishes with payloads exceeding 11 kb.Expanding the payload capacity of transposons could facilitate more sophisticated cargo designs,improving the regulation of expression and minimizing mutagenic risks associated with molecular therapeutics,metabolic engineering,and transgenic animal production.In this study,we improved the Tol2 transposon by increasing protein expression levels using a translational enhancer(QBI SP163,ST)and enhanced the nuclear targeting ability using the nuclear localization protein H2B(SHT).The modified Tol2 and ST transposon efficiently integrated large DNA cargos into human cell cultures(H1299),comparable to the well-established super PiggyBac system.Furthermore,mRNA from ST and SHT showed a significant increase in transgene delivery efficiency of large DNA payloads(8 kb,14 kb,and 24 kb)into zebrafish(Danio rerio).This study presents a modified Tol2 transposon as an enhanced nonviral vector for the delivery of large DNA payloads in transgenic applications.展开更多
Laterality is a crucial physiological process intricately linked to the cilium-centrosome complex during embryo development.Defects in the process can result in severe organ mispositioning.Coiled-coil domain containin...Laterality is a crucial physiological process intricately linked to the cilium-centrosome complex during embryo development.Defects in the process can result in severe organ mispositioning.Coiled-coil domain containing 141(CCDC141)has been previously known as a centrosome-related gene,but its role in left-right(LR)asymmetry has not been characterized.In this study,we utilize the zebrafish model and human exome analysis to elucidate the function of ccdc141 in laterality defects.The knockdown of ccdc141 in zebrafish disrupts early LR signaling pathways,cilia function,and Kupffer's vesicle formation.Unlike ccdc141-knockdown embryos exhibiting aberrant LR patterns,ccdc141-null mutants show no apparent abnormality,suggesting a genetic compensation response effect.In parallel,we observe a marked reduction inα-tubulin acetylation levels in the ccdc141 crispants.The treatment with histone deacetylase(HDAC)inhibitors,particularly the HDAC6 inhibitor,rescues the ccdc141 crispant phenotypes.Furthermore,exome analysis of 70 patients with laterality defects reveals an increased burden of CCDC141 mutations,with in-vivo studies verifying the pathogenicity of the patient mutation CCDC141-R123G.Our findings highlight the critical role of ccdc141 in ciliogenesis and demonstrate that CCDC141 mutations lead to abnormal LR patterns,identifying it as a candidate gene for laterality defects.展开更多
基金supported by the National Science and Technology Innovation 2030 Major Projects(2021ZD0202200)National Natural Science Foundation of China(32171090,81970264)+1 种基金Shanghai Science and Technology Commission(21ZR1482600)2023 Youth Innovation Promotion Association CAS。
文摘Most viruses and transposons serve as effective carriers for the introduction of foreign DNA up to 11 kb into vertebrate genomes.However,their activity markedly diminishes with payloads exceeding 11 kb.Expanding the payload capacity of transposons could facilitate more sophisticated cargo designs,improving the regulation of expression and minimizing mutagenic risks associated with molecular therapeutics,metabolic engineering,and transgenic animal production.In this study,we improved the Tol2 transposon by increasing protein expression levels using a translational enhancer(QBI SP163,ST)and enhanced the nuclear targeting ability using the nuclear localization protein H2B(SHT).The modified Tol2 and ST transposon efficiently integrated large DNA cargos into human cell cultures(H1299),comparable to the well-established super PiggyBac system.Furthermore,mRNA from ST and SHT showed a significant increase in transgene delivery efficiency of large DNA payloads(8 kb,14 kb,and 24 kb)into zebrafish(Danio rerio).This study presents a modified Tol2 transposon as an enhanced nonviral vector for the delivery of large DNA payloads in transgenic applications.
基金supported by the National Natural Science Foundation of China(81970264).
文摘Laterality is a crucial physiological process intricately linked to the cilium-centrosome complex during embryo development.Defects in the process can result in severe organ mispositioning.Coiled-coil domain containing 141(CCDC141)has been previously known as a centrosome-related gene,but its role in left-right(LR)asymmetry has not been characterized.In this study,we utilize the zebrafish model and human exome analysis to elucidate the function of ccdc141 in laterality defects.The knockdown of ccdc141 in zebrafish disrupts early LR signaling pathways,cilia function,and Kupffer's vesicle formation.Unlike ccdc141-knockdown embryos exhibiting aberrant LR patterns,ccdc141-null mutants show no apparent abnormality,suggesting a genetic compensation response effect.In parallel,we observe a marked reduction inα-tubulin acetylation levels in the ccdc141 crispants.The treatment with histone deacetylase(HDAC)inhibitors,particularly the HDAC6 inhibitor,rescues the ccdc141 crispant phenotypes.Furthermore,exome analysis of 70 patients with laterality defects reveals an increased burden of CCDC141 mutations,with in-vivo studies verifying the pathogenicity of the patient mutation CCDC141-R123G.Our findings highlight the critical role of ccdc141 in ciliogenesis and demonstrate that CCDC141 mutations lead to abnormal LR patterns,identifying it as a candidate gene for laterality defects.
