A simple and adaptable method for cloning genes into transposon vectors using topo and restriction systems for chicken embryo transgenesis
Transposon-based genetic transformation enables stable transgene integration in avian genomes and is increasingly used in the development of transgenic chickens for enhanced disease resistance, productivity, and biopharmaceutical applications. Conventional transformation techniques in avian biotechn...
| Main Authors: | , , , , , |
|---|---|
| Format: | Journal Article |
| Language: | Inglés |
| Published: |
Bio-Protocol, LLC
2025
|
| Subjects: | |
| Online Access: | https://hdl.handle.net/10568/176269 |
| _version_ | 1855523782551142400 |
|---|---|
| author | Kirimi, P. Okumu, Noah Maingi, J.M. Ngeranwa, J. Nyaga, P. Binepal, Y. |
| author_browse | Binepal, Y. Kirimi, P. Maingi, J.M. Ngeranwa, J. Nyaga, P. Okumu, Noah |
| author_facet | Kirimi, P. Okumu, Noah Maingi, J.M. Ngeranwa, J. Nyaga, P. Binepal, Y. |
| author_sort | Kirimi, P. |
| collection | Repository of Agricultural Research Outputs (CGSpace) |
| description | Transposon-based genetic transformation enables stable transgene integration in avian genomes and is increasingly used in the development of transgenic chickens for enhanced disease resistance, productivity, and biopharmaceutical applications. Conventional transformation techniques in avian biotechnology, including viral vectors and primordial germ cell (PGC) manipulation, are limited by biosafety risks, low efficiency, and technical complexity. This protocol outlines a two-step cloning approach for generating transposon-compatible gene constructs suitable for chicken embryo microinjection. Topoisomerase-based (TOPO) cloning is used as the first step due to its ability to directly clone PCR-amplified products without the need for restriction site-engineered primers while simultaneously producing an insert flanked with EcoRI restriction sites. The insert is subsequently transferred into the transposon vector through EcoRI-mediated restriction digestion and ligation. This approach simplifies construct generation by integrating the speed of TOPO cloning with the precision of restriction cloning, while ensuring compatibility with transposon-mediated integration systems. The protocol is efficient, reproducible, and does not require specialized equipment, providing a practical and scalable tool for gene construct assembly in avian transgenesis research. |
| format | Journal Article |
| id | CGSpace176269 |
| institution | CGIAR Consortium |
| language | Inglés |
| publishDate | 2025 |
| publishDateRange | 2025 |
| publishDateSort | 2025 |
| publisher | Bio-Protocol, LLC |
| publisherStr | Bio-Protocol, LLC |
| record_format | dspace |
| spelling | CGSpace1762692025-12-08T10:29:22Z A simple and adaptable method for cloning genes into transposon vectors using topo and restriction systems for chicken embryo transgenesis Kirimi, P. Okumu, Noah Maingi, J.M. Ngeranwa, J. Nyaga, P. Binepal, Y. chickens genetics protocols Transposon-based genetic transformation enables stable transgene integration in avian genomes and is increasingly used in the development of transgenic chickens for enhanced disease resistance, productivity, and biopharmaceutical applications. Conventional transformation techniques in avian biotechnology, including viral vectors and primordial germ cell (PGC) manipulation, are limited by biosafety risks, low efficiency, and technical complexity. This protocol outlines a two-step cloning approach for generating transposon-compatible gene constructs suitable for chicken embryo microinjection. Topoisomerase-based (TOPO) cloning is used as the first step due to its ability to directly clone PCR-amplified products without the need for restriction site-engineered primers while simultaneously producing an insert flanked with EcoRI restriction sites. The insert is subsequently transferred into the transposon vector through EcoRI-mediated restriction digestion and ligation. This approach simplifies construct generation by integrating the speed of TOPO cloning with the precision of restriction cloning, while ensuring compatibility with transposon-mediated integration systems. The protocol is efficient, reproducible, and does not require specialized equipment, providing a practical and scalable tool for gene construct assembly in avian transgenesis research. 2025 2025-09-01T09:20:30Z 2025-09-01T09:20:30Z Journal Article https://hdl.handle.net/10568/176269 en Open Access Bio-Protocol, LLC Kirimi, P., Okumu, N., Maingi, J.M., Ngeranwa, J., Nyaga, P. and Binepal, Y. 2025. A simple and adaptable method for cloning genes into transposon vectors using topo and restriction systems for chicken embryo transgenesis. Bio-protocol 15(16): e5416. |
| spellingShingle | chickens genetics protocols Kirimi, P. Okumu, Noah Maingi, J.M. Ngeranwa, J. Nyaga, P. Binepal, Y. A simple and adaptable method for cloning genes into transposon vectors using topo and restriction systems for chicken embryo transgenesis |
| title | A simple and adaptable method for cloning genes into transposon vectors using topo and restriction systems for chicken embryo transgenesis |
| title_full | A simple and adaptable method for cloning genes into transposon vectors using topo and restriction systems for chicken embryo transgenesis |
| title_fullStr | A simple and adaptable method for cloning genes into transposon vectors using topo and restriction systems for chicken embryo transgenesis |
| title_full_unstemmed | A simple and adaptable method for cloning genes into transposon vectors using topo and restriction systems for chicken embryo transgenesis |
| title_short | A simple and adaptable method for cloning genes into transposon vectors using topo and restriction systems for chicken embryo transgenesis |
| title_sort | simple and adaptable method for cloning genes into transposon vectors using topo and restriction systems for chicken embryo transgenesis |
| topic | chickens genetics protocols |
| url | https://hdl.handle.net/10568/176269 |
| work_keys_str_mv | AT kirimip asimpleandadaptablemethodforcloninggenesintotransposonvectorsusingtopoandrestrictionsystemsforchickenembryotransgenesis AT okumunoah asimpleandadaptablemethodforcloninggenesintotransposonvectorsusingtopoandrestrictionsystemsforchickenembryotransgenesis AT maingijm asimpleandadaptablemethodforcloninggenesintotransposonvectorsusingtopoandrestrictionsystemsforchickenembryotransgenesis AT ngeranwaj asimpleandadaptablemethodforcloninggenesintotransposonvectorsusingtopoandrestrictionsystemsforchickenembryotransgenesis AT nyagap asimpleandadaptablemethodforcloninggenesintotransposonvectorsusingtopoandrestrictionsystemsforchickenembryotransgenesis AT binepaly asimpleandadaptablemethodforcloninggenesintotransposonvectorsusingtopoandrestrictionsystemsforchickenembryotransgenesis AT kirimip simpleandadaptablemethodforcloninggenesintotransposonvectorsusingtopoandrestrictionsystemsforchickenembryotransgenesis AT okumunoah simpleandadaptablemethodforcloninggenesintotransposonvectorsusingtopoandrestrictionsystemsforchickenembryotransgenesis AT maingijm simpleandadaptablemethodforcloninggenesintotransposonvectorsusingtopoandrestrictionsystemsforchickenembryotransgenesis AT ngeranwaj simpleandadaptablemethodforcloninggenesintotransposonvectorsusingtopoandrestrictionsystemsforchickenembryotransgenesis AT nyagap simpleandadaptablemethodforcloninggenesintotransposonvectorsusingtopoandrestrictionsystemsforchickenembryotransgenesis AT binepaly simpleandadaptablemethodforcloninggenesintotransposonvectorsusingtopoandrestrictionsystemsforchickenembryotransgenesis |