The axolotl is broadly used in regenerative, developmental, and evolutionary biology research. Targeted gene knock-in is crucial for precision transgenesis, enabling disease modeling, visualization, tracking, and func...The axolotl is broadly used in regenerative, developmental, and evolutionary biology research. Targeted gene knock-in is crucial for precision transgenesis, enabling disease modeling, visualization, tracking, and functional manipulation of specific cells or genes of interest(GOIs). Existing CRISPR/Cas9-mediated homology-independent method for gene knock-in often causes “scars/indels” at integration junctions.Here, we develop a CRISPR/Cas9-mediated semi-homology-directed recombination(HDR) knock-in method using a donor construct containing a single homology arm for the precise integration of GOIs.This semi-HDR approach achieves seamless single-end integration of the Cherry reporter gene and a large inducible Cre cassette into intronless genes like Sox2 and Neurod6 in axolotls, which are challenging to modify with the homology-independent method. Additionally, we integrate the inducible Cre cassette into intron-containing loci(e.g., Nkx2.2 and FoxA2) without introducing indels via semi-HDR. GOIs are properly expressed in F0 founders, with approximately 5%-10% showing precise integration confirmed by genotyping. Furthermore, using the Nkx2.2:CreER^(T2)line, we fate-map spinal cord p3 neural progenitor cells,revealing that Nkx2.2^(+) cells adopt different lineages in development and regeneration, preferentially generating motoneurons over oligodendrocytes during regeneration. Overall, this semi-HDR method balances efficiency and precision in the integration of GOIs, providing a valuable tool for generating knock-in axolotls and potentially extending to other species.展开更多
Although Sirt2 is primarily expressed in oligodendrocytes of the central nervous system,its role in oligodendroglial lineage differentiation is not fully understood.Our findings demonstrate that the transcription fac...Although Sirt2 is primarily expressed in oligodendrocytes of the central nervous system,its role in oligodendroglial lineage differentiation is not fully understood.Our findings demonstrate that the transcription factor Nkx2.2 binds to the Sirt2 promoter via histone deacetylase 1(HDAC-1),the binding site for Nkx2.2 maps close to the start codon of the Sirt2 gene,and Nkx2.2 negatively regulates Sirt2 expression in CG4 cells,an oligodendroglial precursor cell line.HDAC-1 knock-down not only significantly attenuates the binding capacity of Nkx2.2 to the Sirt2 promoter but also releases repression of Sirt2 expression by Nkx2.2.Nkx2.2 overexpression down-regulates Sirt2 expression and delays differentiation of CG4 cells;in contrast,up-regulation of Sirt2 does not impact Nkx2.2 expression level.Sirt2 knock-down via RNAi or inhibition of Sirt2 by sirtinol,a Sirt2 activity inhibitor,blocks CG4 cell differentiation.Over-expression of Sirt2 facilitates CG4 cell differentiation at both molecular and cellular levels,enhancing expression of myelin basic protein and facilitating the growth of cell processes.We have conclusively demonstrated that Sirt2 enhances CG4 oligodendroglial differentiation and report a novel mechanism through which Nkx2.2 represses CG4 oligodendroglial differentiation via Sirt2.展开更多
Our previous study revealed that intragastric administration of naringin improved remyelination in rats with spinal cord injury and promoted the recovery of neurological function of the injured spinal cord.This study ...Our previous study revealed that intragastric administration of naringin improved remyelination in rats with spinal cord injury and promoted the recovery of neurological function of the injured spinal cord.This study sought to reveal the mechanisms by which naringin improves oligodendrocyte precursor cell differentiation and maturation,and promotes remyelination.Spinal cord injury was induced in rats by the weight-drop method.Naringin was intragastrically administered daily（20,40 mg/kg） for 4 weeks after spinal cord injury induction.Behavioral assessment,histopathological staining,immunofluorescence spectroscopy,ultrastructural analysis and biochemical assays were employed.Naringin treatment remarkably mitigated demyelination in the white matter,increased the quality of myelinated nerve fibers and myelin sheath thickness,promoted oligodendrocyte precursor cell differentiation by upregulating the expression of NKx2.2 and 2′3′-cyclic nucleotide 3′-phosphodiesterase,and inhibited β-catenin expression and glycogen synthase kinase-3β（GSK-3β） phosphorylation.These findings indicate that naringin treatment regulates oligodendrocyte precursor cell differentiation and promotes remyelination after spinal cord injury through the β-catenin/GSK-3β signaling pathway.展开更多
基金supported by the National Key R&D Program of China(2021YFA0805000,2023YFA1800600,2019YFE0106700)the National Natural Science Foundation of China(92268114,31970782,32070819)+1 种基金the High-level Hospital Construction Project of GuangdongProvincial People'sHospital(DFJHBF202103 and KJ012021012)BGI grant(BGIRSZ20210002).
文摘The axolotl is broadly used in regenerative, developmental, and evolutionary biology research. Targeted gene knock-in is crucial for precision transgenesis, enabling disease modeling, visualization, tracking, and functional manipulation of specific cells or genes of interest(GOIs). Existing CRISPR/Cas9-mediated homology-independent method for gene knock-in often causes “scars/indels” at integration junctions.Here, we develop a CRISPR/Cas9-mediated semi-homology-directed recombination(HDR) knock-in method using a donor construct containing a single homology arm for the precise integration of GOIs.This semi-HDR approach achieves seamless single-end integration of the Cherry reporter gene and a large inducible Cre cassette into intronless genes like Sox2 and Neurod6 in axolotls, which are challenging to modify with the homology-independent method. Additionally, we integrate the inducible Cre cassette into intron-containing loci(e.g., Nkx2.2 and FoxA2) without introducing indels via semi-HDR. GOIs are properly expressed in F0 founders, with approximately 5%-10% showing precise integration confirmed by genotyping. Furthermore, using the Nkx2.2:CreER^(T2)line, we fate-map spinal cord p3 neural progenitor cells,revealing that Nkx2.2^(+) cells adopt different lineages in development and regeneration, preferentially generating motoneurons over oligodendrocytes during regeneration. Overall, this semi-HDR method balances efficiency and precision in the integration of GOIs, providing a valuable tool for generating knock-in axolotls and potentially extending to other species.
文摘Although Sirt2 is primarily expressed in oligodendrocytes of the central nervous system,its role in oligodendroglial lineage differentiation is not fully understood.Our findings demonstrate that the transcription factor Nkx2.2 binds to the Sirt2 promoter via histone deacetylase 1(HDAC-1),the binding site for Nkx2.2 maps close to the start codon of the Sirt2 gene,and Nkx2.2 negatively regulates Sirt2 expression in CG4 cells,an oligodendroglial precursor cell line.HDAC-1 knock-down not only significantly attenuates the binding capacity of Nkx2.2 to the Sirt2 promoter but also releases repression of Sirt2 expression by Nkx2.2.Nkx2.2 overexpression down-regulates Sirt2 expression and delays differentiation of CG4 cells;in contrast,up-regulation of Sirt2 does not impact Nkx2.2 expression level.Sirt2 knock-down via RNAi or inhibition of Sirt2 by sirtinol,a Sirt2 activity inhibitor,blocks CG4 cell differentiation.Over-expression of Sirt2 facilitates CG4 cell differentiation at both molecular and cellular levels,enhancing expression of myelin basic protein and facilitating the growth of cell processes.We have conclusively demonstrated that Sirt2 enhances CG4 oligodendroglial differentiation and report a novel mechanism through which Nkx2.2 represses CG4 oligodendroglial differentiation via Sirt2.
基金supported by the Natural Science Foundation of Beijing of China,No.7164317the Beijing Tsinghua Changgung Hospital Fund,No.12015C1028the National Natural Science Foundation of China,No.31400717
文摘Our previous study revealed that intragastric administration of naringin improved remyelination in rats with spinal cord injury and promoted the recovery of neurological function of the injured spinal cord.This study sought to reveal the mechanisms by which naringin improves oligodendrocyte precursor cell differentiation and maturation,and promotes remyelination.Spinal cord injury was induced in rats by the weight-drop method.Naringin was intragastrically administered daily（20,40 mg/kg） for 4 weeks after spinal cord injury induction.Behavioral assessment,histopathological staining,immunofluorescence spectroscopy,ultrastructural analysis and biochemical assays were employed.Naringin treatment remarkably mitigated demyelination in the white matter,increased the quality of myelinated nerve fibers and myelin sheath thickness,promoted oligodendrocyte precursor cell differentiation by upregulating the expression of NKx2.2 and 2′3′-cyclic nucleotide 3′-phosphodiesterase,and inhibited β-catenin expression and glycogen synthase kinase-3β（GSK-3β） phosphorylation.These findings indicate that naringin treatment regulates oligodendrocyte precursor cell differentiation and promotes remyelination after spinal cord injury through the β-catenin/GSK-3β signaling pathway.
