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
Desmin and vimentin are major components of intermediate filament proteins in cardiac myocytes. We developed a primary cell culture method for cardiac myocytes of axolotl embryos. Cardiac myocytes of embryonic stage 3...Desmin and vimentin are major components of intermediate filament proteins in cardiac myocytes. We developed a primary cell culture method for cardiac myocytes of axolotl embryos. Cardiac myocytes of embryonic stage 39 were cultured for 1-14 days. Myocytes showed spontaneous contractions (15-30 beats/min) after 48-72 hours in culture, round shape and large irregular projections. Desmin and vimentin were observed in the cultured myocytes by means of immunofluorescent staining in combination with immunofluorescent microscopy. Immunofluorescent staining of the cultured cardiac myocytes after different lengths of time in culture(3,6,9 days) showed that vimentin staining was stronger than desmin staining during the early stages of culture (3 days). The myocytes exhibited various forms of staining, including parallel lines and interconnected networks. Some lines showed regular striation; most of the myofibrils were arranged in parallel arrays along the cell's long axis. Both desmin and vimentin in the cell appeared to encirele the Z lines and to link myofibrils laterally at the Z lines.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Desmin and vimentin are major components of intermediate filament proteins in cardiac myocytes. We developed a primary cell culture method for cardiac myocytes of axolotl embryos. Cardiac myocytes of embryonic stage 39 were cultured for 1-14 days. Myocytes showed spontaneous contractions (15-30 beats/min) after 48-72 hours in culture, round shape and large irregular projections. Desmin and vimentin were observed in the cultured myocytes by means of immunofluorescent staining in combination with immunofluorescent microscopy. Immunofluorescent staining of the cultured cardiac myocytes after different lengths of time in culture(3,6,9 days) showed that vimentin staining was stronger than desmin staining during the early stages of culture (3 days). The myocytes exhibited various forms of staining, including parallel lines and interconnected networks. Some lines showed regular striation; most of the myofibrils were arranged in parallel arrays along the cell's long axis. Both desmin and vimentin in the cell appeared to encirele the Z lines and to link myofibrils laterally at the Z lines.
