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
Starting from the importance of preserving our information and protecting our devices against attacks on their vulnerabilities, this article aims to establish the level of knowledge in computer security and problem-so...Starting from the importance of preserving our information and protecting our devices against attacks on their vulnerabilities, this article aims to establish the level of knowledge in computer security and problem-solving of students at a university in northwest Mexico. This research has a quantitative approach where the European Framework of Digital Competencies for Citizenship was used as a reference to identify and establish the level of the competencies that were evaluated. The IKANOS Test was used as a data collection tool. The results show that students know the importance of keeping their devices safe and how valuable the information found on them is. On the other hand, the results also show a considerable percentage of students who do not have the knowledge and are at a basic level of knowledge to solve technical problems with their devices.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Starting from the importance of preserving our information and protecting our devices against attacks on their vulnerabilities, this article aims to establish the level of knowledge in computer security and problem-solving of students at a university in northwest Mexico. This research has a quantitative approach where the European Framework of Digital Competencies for Citizenship was used as a reference to identify and establish the level of the competencies that were evaluated. The IKANOS Test was used as a data collection tool. The results show that students know the importance of keeping their devices safe and how valuable the information found on them is. On the other hand, the results also show a considerable percentage of students who do not have the knowledge and are at a basic level of knowledge to solve technical problems with their devices.
