Converting monoclonal antibodies into Fab fragments for transient expression in mammalian cells.
In this chapter, protocols are described for converting mouse monoclonal antibodies into recombinant Fabs for transient expression in mammalian cells. Variable region genes are cloned by reverse transcription: PCR using either sequence specific or mixed 5' primers that hybridise to the first fr...
| Main Authors: | , , , , |
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| Format: | Journal article |
| Language: | English |
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2012
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| _version_ | 1797065011202359296 |
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| author | Nettleship, J Flanagan, A Rahman-Huq, N Hamer, R Owens, R |
| author_facet | Nettleship, J Flanagan, A Rahman-Huq, N Hamer, R Owens, R |
| author_sort | Nettleship, J |
| collection | OXFORD |
| description | In this chapter, protocols are described for converting mouse monoclonal antibodies into recombinant Fabs for transient expression in mammalian cells. Variable region genes are cloned by reverse transcription: PCR using either sequence specific or mixed 5' primers that hybridise to the first framework sequence of the mouse light and heavy chains and 3' primers that bind to the heavy- and light-chain constant regions. The amplified sequences are inserted into mammalian cell expression vectors by In-Fusion™ cloning. This method allows vector and amplified DNA sequences to be seamlessly joined in a ligation-independent reaction. Transient co-expression of light-chain and heavy-chain genes in HEK 293T cells enables production of recombinant Fabs for functional and structural studies. |
| first_indexed | 2024-03-06T21:22:31Z |
| format | Journal article |
| id | oxford-uuid:41f3ce5e-3a07-47bb-8987-a7d0c84bf9cb |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T21:22:31Z |
| publishDate | 2012 |
| record_format | dspace |
| spelling | oxford-uuid:41f3ce5e-3a07-47bb-8987-a7d0c84bf9cb2022-03-26T14:46:39ZConverting monoclonal antibodies into Fab fragments for transient expression in mammalian cells.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:41f3ce5e-3a07-47bb-8987-a7d0c84bf9cbEnglishSymplectic Elements at Oxford2012Nettleship, JFlanagan, ARahman-Huq, NHamer, ROwens, RIn this chapter, protocols are described for converting mouse monoclonal antibodies into recombinant Fabs for transient expression in mammalian cells. Variable region genes are cloned by reverse transcription: PCR using either sequence specific or mixed 5' primers that hybridise to the first framework sequence of the mouse light and heavy chains and 3' primers that bind to the heavy- and light-chain constant regions. The amplified sequences are inserted into mammalian cell expression vectors by In-Fusion™ cloning. This method allows vector and amplified DNA sequences to be seamlessly joined in a ligation-independent reaction. Transient co-expression of light-chain and heavy-chain genes in HEK 293T cells enables production of recombinant Fabs for functional and structural studies. |
| spellingShingle | Nettleship, J Flanagan, A Rahman-Huq, N Hamer, R Owens, R Converting monoclonal antibodies into Fab fragments for transient expression in mammalian cells. |
| title | Converting monoclonal antibodies into Fab fragments for transient expression in mammalian cells. |
| title_full | Converting monoclonal antibodies into Fab fragments for transient expression in mammalian cells. |
| title_fullStr | Converting monoclonal antibodies into Fab fragments for transient expression in mammalian cells. |
| title_full_unstemmed | Converting monoclonal antibodies into Fab fragments for transient expression in mammalian cells. |
| title_short | Converting monoclonal antibodies into Fab fragments for transient expression in mammalian cells. |
| title_sort | converting monoclonal antibodies into fab fragments for transient expression in mammalian cells |
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