Single/low-copy integration of transgenes in <it>Caenorhabditis elegans </it>using an ultraviolet trimethylpsoralen method
<p>Abstract</p> <p>Background</p> <p>Transgenic strains of <it>Caenorhabditis elegans </it>are typically generated by injecting DNA into the germline to form multi-copy extrachromosomal arrays. These transgenes are semi-stable and their expression is silence...
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| Format: | Article |
| Language: | English |
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BMC
2012-01-01
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| Series: | BMC Biotechnology |
| Online Access: | http://www.biomedcentral.com/1472-6750/12/1 |
| _version_ | 1811286512390635520 |
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| author | Kage-Nakadai Eriko Kobuna Hiroyuki Funatsu Osamu Otori Muneyoshi Gengyo-Ando Keiko Yoshina Sawako Hori Sayaka Mitani Shohei |
| author_facet | Kage-Nakadai Eriko Kobuna Hiroyuki Funatsu Osamu Otori Muneyoshi Gengyo-Ando Keiko Yoshina Sawako Hori Sayaka Mitani Shohei |
| author_sort | Kage-Nakadai Eriko |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Transgenic strains of <it>Caenorhabditis elegans </it>are typically generated by injecting DNA into the germline to form multi-copy extrachromosomal arrays. These transgenes are semi-stable and their expression is silenced in the germline. Mos1 transposon or microparticle bombardment methods have been developed to create single- or low-copy chromosomal integrated lines. Here we report an alternative method using ultraviolet trimethylpsoralen (UV/TMP) to generate single/low-copy gene integrations.</p> <p>Results</p> <p>We successfully integrated low-copy transgenes from extrachromosomal arrays using positive selection based on temperature sensitivity with a <it>vps-45 </it>rescue fragment and negative selection based on benzimidazole sensitivity with a <it>ben-1 </it>rescue fragment. We confirmed that the integrants express transgenes in the germline. Quantitative PCR revealed that strains generated by this method contain single- or low-copy transgenes. Moreover, positive selection marker genes flanked by LoxP sites were excised by Cre recombinase mRNA microinjection, demonstrating Cre-mediated chromosomal excision for the first time in <it>C. elegans</it>.</p> <p>Conclusion</p> <p>Our UV/TMP integration method, based on familiar extrachromosomal transgenics, provides a useful approach for generating single/low-copy gene integrations.</p> |
| first_indexed | 2024-04-13T03:01:18Z |
| format | Article |
| id | doaj.art-bd25592f3c574d038038e09a73017454 |
| institution | Directory Open Access Journal |
| issn | 1472-6750 |
| language | English |
| last_indexed | 2024-04-13T03:01:18Z |
| publishDate | 2012-01-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biotechnology |
| spelling | doaj.art-bd25592f3c574d038038e09a730174542022-12-22T03:05:26ZengBMCBMC Biotechnology1472-67502012-01-01121110.1186/1472-6750-12-1Single/low-copy integration of transgenes in <it>Caenorhabditis elegans </it>using an ultraviolet trimethylpsoralen methodKage-Nakadai ErikoKobuna HiroyukiFunatsu OsamuOtori MuneyoshiGengyo-Ando KeikoYoshina SawakoHori SayakaMitani Shohei<p>Abstract</p> <p>Background</p> <p>Transgenic strains of <it>Caenorhabditis elegans </it>are typically generated by injecting DNA into the germline to form multi-copy extrachromosomal arrays. These transgenes are semi-stable and their expression is silenced in the germline. Mos1 transposon or microparticle bombardment methods have been developed to create single- or low-copy chromosomal integrated lines. Here we report an alternative method using ultraviolet trimethylpsoralen (UV/TMP) to generate single/low-copy gene integrations.</p> <p>Results</p> <p>We successfully integrated low-copy transgenes from extrachromosomal arrays using positive selection based on temperature sensitivity with a <it>vps-45 </it>rescue fragment and negative selection based on benzimidazole sensitivity with a <it>ben-1 </it>rescue fragment. We confirmed that the integrants express transgenes in the germline. Quantitative PCR revealed that strains generated by this method contain single- or low-copy transgenes. Moreover, positive selection marker genes flanked by LoxP sites were excised by Cre recombinase mRNA microinjection, demonstrating Cre-mediated chromosomal excision for the first time in <it>C. elegans</it>.</p> <p>Conclusion</p> <p>Our UV/TMP integration method, based on familiar extrachromosomal transgenics, provides a useful approach for generating single/low-copy gene integrations.</p>http://www.biomedcentral.com/1472-6750/12/1 |
| spellingShingle | Kage-Nakadai Eriko Kobuna Hiroyuki Funatsu Osamu Otori Muneyoshi Gengyo-Ando Keiko Yoshina Sawako Hori Sayaka Mitani Shohei Single/low-copy integration of transgenes in <it>Caenorhabditis elegans </it>using an ultraviolet trimethylpsoralen method BMC Biotechnology |
| title | Single/low-copy integration of transgenes in <it>Caenorhabditis elegans </it>using an ultraviolet trimethylpsoralen method |
| title_full | Single/low-copy integration of transgenes in <it>Caenorhabditis elegans </it>using an ultraviolet trimethylpsoralen method |
| title_fullStr | Single/low-copy integration of transgenes in <it>Caenorhabditis elegans </it>using an ultraviolet trimethylpsoralen method |
| title_full_unstemmed | Single/low-copy integration of transgenes in <it>Caenorhabditis elegans </it>using an ultraviolet trimethylpsoralen method |
| title_short | Single/low-copy integration of transgenes in <it>Caenorhabditis elegans </it>using an ultraviolet trimethylpsoralen method |
| title_sort | single low copy integration of transgenes in it caenorhabditis elegans it using an ultraviolet trimethylpsoralen method |
| url | http://www.biomedcentral.com/1472-6750/12/1 |
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