Development of a new strategy for inducible and reversible gene knockout.
The ability to inactivate gene function has contributed to functional genetics studies. Current techniques of gene knockout using Cre-loxP recombination and RNAi have achieved varying degrees of success despite certain limitations. To overcome these limitations, we initiated the development...
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| Other Authors: | |
| Format: | Final Year Project (FYP) |
| Language: | English |
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2013
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| Online Access: | http://hdl.handle.net/10356/53790 |
| _version_ | 1811685037804879872 |
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| author | Tan, Hanrong. |
| author2 | Klaus Erik Karjalainen |
| author_facet | Klaus Erik Karjalainen Tan, Hanrong. |
| author_sort | Tan, Hanrong. |
| collection | NTU |
| description | The ability to inactivate gene function has contributed to functional genetics studies. Current
techniques of gene knockout using Cre-loxP recombination and RNAi have achieved varying
degrees of success despite certain limitations. To overcome these limitations, we initiated the
development of a new strategy to achieve inducible and reversible gene knockout. The
strategy requires two components, a tetracycline inducible repressor (TetR-kid) and a
repressor binding sequence (ΔTRE). TetR-kid was obtained by coupling the Tetracycline
repressor protein (TetR) to the Kid1 protein. ΔTRE is a modified tetracycline response
element (TRE). Here, a Rosa26 targeting vector was constructed in efforts of developing this
strategy. The vector was designed to introduce the TetR-kid transgene into the Rosa26
genetic locus to achieve ubiquitous and uniform expression of TetR-kid. The vector was
obtained by modifying a BAC containing the locus. By recombineering, the TetR-kid
transgene along with an ACN cassette for positive selection was introduced into the locus
followed by a second recombineering step to retrieve the modified BAC fragment into a
vector that contains a negative selection marker, a diphtheria toxin fragment a (DTA) gene.
This targeting vector would be used for gene targeting in ES cells in efforts to generate TetRkid
knock-in mouse. |
| first_indexed | 2024-10-01T04:38:09Z |
| format | Final Year Project (FYP) |
| id | ntu-10356/53790 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T04:38:09Z |
| publishDate | 2013 |
| record_format | dspace |
| spelling | ntu-10356/537902023-02-28T18:01:51Z Development of a new strategy for inducible and reversible gene knockout. Tan, Hanrong. Klaus Erik Karjalainen School of Biological Sciences DRNTU::Science::Biological sciences::Molecular biology The ability to inactivate gene function has contributed to functional genetics studies. Current techniques of gene knockout using Cre-loxP recombination and RNAi have achieved varying degrees of success despite certain limitations. To overcome these limitations, we initiated the development of a new strategy to achieve inducible and reversible gene knockout. The strategy requires two components, a tetracycline inducible repressor (TetR-kid) and a repressor binding sequence (ΔTRE). TetR-kid was obtained by coupling the Tetracycline repressor protein (TetR) to the Kid1 protein. ΔTRE is a modified tetracycline response element (TRE). Here, a Rosa26 targeting vector was constructed in efforts of developing this strategy. The vector was designed to introduce the TetR-kid transgene into the Rosa26 genetic locus to achieve ubiquitous and uniform expression of TetR-kid. The vector was obtained by modifying a BAC containing the locus. By recombineering, the TetR-kid transgene along with an ACN cassette for positive selection was introduced into the locus followed by a second recombineering step to retrieve the modified BAC fragment into a vector that contains a negative selection marker, a diphtheria toxin fragment a (DTA) gene. This targeting vector would be used for gene targeting in ES cells in efforts to generate TetRkid knock-in mouse. Bachelor of Science in Biological Sciences 2013-06-07T06:27:21Z 2013-06-07T06:27:21Z 2013 2013 Final Year Project (FYP) http://hdl.handle.net/10356/53790 en Nanyang Technological University 29 p. application/pdf |
| spellingShingle | DRNTU::Science::Biological sciences::Molecular biology Tan, Hanrong. Development of a new strategy for inducible and reversible gene knockout. |
| title | Development of a new strategy for inducible and reversible gene knockout. |
| title_full | Development of a new strategy for inducible and reversible gene knockout. |
| title_fullStr | Development of a new strategy for inducible and reversible gene knockout. |
| title_full_unstemmed | Development of a new strategy for inducible and reversible gene knockout. |
| title_short | Development of a new strategy for inducible and reversible gene knockout. |
| title_sort | development of a new strategy for inducible and reversible gene knockout |
| topic | DRNTU::Science::Biological sciences::Molecular biology |
| url | http://hdl.handle.net/10356/53790 |
| work_keys_str_mv | AT tanhanrong developmentofanewstrategyforinducibleandreversiblegeneknockout |