Retrofitting BACs with G418 resistance, luciferase, and <it>oriP </it>and <it>EBNA-1 </it>– new vectors for <it>in vitro </it>and <it>in vivo </it>delivery
<p>Abstract</p> <p>Background</p> <p>Bacterial artificial chromosomes (BACs) have been used extensively for sequencing the human and mouse genomes and are thus readily available for most genes. The large size of BACs means that they can generally carry intact genes with...
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| Format: | Article |
| Language: | English |
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BMC
2003-02-01
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| Series: | BMC Biotechnology |
| Online Access: | http://www.biomedcentral.com/1472-6750/3/2 |
| _version_ | 1818484132528783360 |
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| author | Wagner Ernst D'Aiuto Leonardo Kotzamanis George Magin-Lachmann Christine Huxley Clare |
| author_facet | Wagner Ernst D'Aiuto Leonardo Kotzamanis George Magin-Lachmann Christine Huxley Clare |
| author_sort | Wagner Ernst |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Bacterial artificial chromosomes (BACs) have been used extensively for sequencing the human and mouse genomes and are thus readily available for most genes. The large size of BACs means that they can generally carry intact genes with all the long range controlling elements that drive full levels of tissue-specific expression. For gene expression studies and gene therapy applications it is useful to be able to retrofit the BACs with selectable genes such as G418 resistance, reporter genes such as luciferase, and <it>oriP/EBNA-1 </it>from Epstein Barr virus which allows long term episomal maintenance in mammalian cells.</p> <p>Results</p> <p>We describe a series of retrofitting plasmids and a protocol for <it>in vivo loxP</it>/Cre recombination. The vector pRetroNeo carries a G418 resistance cassette, pRetroNeoLuc carries G418 resistance and a luciferase expression cassette, pRetroNeoLucOE carries G418 resistance, luciferase and an <it>oriP/EBNA-1 </it>cassette and pRetroNeoOE carries G418 resistance and <it>oriP/EBNA-1</it>. These vectors can be efficiently retrofitted onto BACs without rearrangement of the BAC clone. The luciferase cassette is expressed efficiently from the retrofitting plasmids and from retrofitted BACs after transient transfection of B16F10 cells in tissue culture and after electroporation into muscles of BALB/c mice <it>in vivo</it>. We also show that a BAC carrying GFP, <it>oriP </it>and <it>EBNA-1 </it>can be transfected into B16F10 cells with Lipofectamine 2000 and can be rescued intact after 5 weeks.</p> <p>Conclusion</p> <p>The pRetro vectors allow efficient retrofitting of BACs with G418 resistance, luciferase and/or <it>oriP/EBNA-1 </it>using <it>in vivo </it>expression of Cre. The luciferase reporter gene is expressed after transient transfection of retrofitted BACs into cells in tissue culture and after electroporation into mouse muscle <it>in vivo</it>. <it>OriP/EBNA-1 </it>allows stable maintenance of a 150-kb BAC without rearrangement for at least 5 weeks.</p> |
| first_indexed | 2024-12-10T15:51:08Z |
| format | Article |
| id | doaj.art-eeac2e86524e40b6a911de8fbb778db4 |
| institution | Directory Open Access Journal |
| issn | 1472-6750 |
| language | English |
| last_indexed | 2024-12-10T15:51:08Z |
| publishDate | 2003-02-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biotechnology |
| spelling | doaj.art-eeac2e86524e40b6a911de8fbb778db42022-12-22T01:42:49ZengBMCBMC Biotechnology1472-67502003-02-0131210.1186/1472-6750-3-2Retrofitting BACs with G418 resistance, luciferase, and <it>oriP </it>and <it>EBNA-1 </it>– new vectors for <it>in vitro </it>and <it>in vivo </it>deliveryWagner ErnstD'Aiuto LeonardoKotzamanis GeorgeMagin-Lachmann ChristineHuxley Clare<p>Abstract</p> <p>Background</p> <p>Bacterial artificial chromosomes (BACs) have been used extensively for sequencing the human and mouse genomes and are thus readily available for most genes. The large size of BACs means that they can generally carry intact genes with all the long range controlling elements that drive full levels of tissue-specific expression. For gene expression studies and gene therapy applications it is useful to be able to retrofit the BACs with selectable genes such as G418 resistance, reporter genes such as luciferase, and <it>oriP/EBNA-1 </it>from Epstein Barr virus which allows long term episomal maintenance in mammalian cells.</p> <p>Results</p> <p>We describe a series of retrofitting plasmids and a protocol for <it>in vivo loxP</it>/Cre recombination. The vector pRetroNeo carries a G418 resistance cassette, pRetroNeoLuc carries G418 resistance and a luciferase expression cassette, pRetroNeoLucOE carries G418 resistance, luciferase and an <it>oriP/EBNA-1 </it>cassette and pRetroNeoOE carries G418 resistance and <it>oriP/EBNA-1</it>. These vectors can be efficiently retrofitted onto BACs without rearrangement of the BAC clone. The luciferase cassette is expressed efficiently from the retrofitting plasmids and from retrofitted BACs after transient transfection of B16F10 cells in tissue culture and after electroporation into muscles of BALB/c mice <it>in vivo</it>. We also show that a BAC carrying GFP, <it>oriP </it>and <it>EBNA-1 </it>can be transfected into B16F10 cells with Lipofectamine 2000 and can be rescued intact after 5 weeks.</p> <p>Conclusion</p> <p>The pRetro vectors allow efficient retrofitting of BACs with G418 resistance, luciferase and/or <it>oriP/EBNA-1 </it>using <it>in vivo </it>expression of Cre. The luciferase reporter gene is expressed after transient transfection of retrofitted BACs into cells in tissue culture and after electroporation into mouse muscle <it>in vivo</it>. <it>OriP/EBNA-1 </it>allows stable maintenance of a 150-kb BAC without rearrangement for at least 5 weeks.</p>http://www.biomedcentral.com/1472-6750/3/2 |
| spellingShingle | Wagner Ernst D'Aiuto Leonardo Kotzamanis George Magin-Lachmann Christine Huxley Clare Retrofitting BACs with G418 resistance, luciferase, and <it>oriP </it>and <it>EBNA-1 </it>– new vectors for <it>in vitro </it>and <it>in vivo </it>delivery BMC Biotechnology |
| title | Retrofitting BACs with G418 resistance, luciferase, and <it>oriP </it>and <it>EBNA-1 </it>– new vectors for <it>in vitro </it>and <it>in vivo </it>delivery |
| title_full | Retrofitting BACs with G418 resistance, luciferase, and <it>oriP </it>and <it>EBNA-1 </it>– new vectors for <it>in vitro </it>and <it>in vivo </it>delivery |
| title_fullStr | Retrofitting BACs with G418 resistance, luciferase, and <it>oriP </it>and <it>EBNA-1 </it>– new vectors for <it>in vitro </it>and <it>in vivo </it>delivery |
| title_full_unstemmed | Retrofitting BACs with G418 resistance, luciferase, and <it>oriP </it>and <it>EBNA-1 </it>– new vectors for <it>in vitro </it>and <it>in vivo </it>delivery |
| title_short | Retrofitting BACs with G418 resistance, luciferase, and <it>oriP </it>and <it>EBNA-1 </it>– new vectors for <it>in vitro </it>and <it>in vivo </it>delivery |
| title_sort | retrofitting bacs with g418 resistance luciferase and it orip it and it ebna 1 it new vectors for it in vitro it and it in vivo it delivery |
| url | http://www.biomedcentral.com/1472-6750/3/2 |
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