Construction and characterization of a novel miniaturized filamentous phagemid for targeted mammalian gene transfer
Abstract Background As simplistic proteinaceous carriers of genetic material, phages offer great potential as targeted vectors for mammalian transgene delivery. The filamentous phage M13 is a single-stranded DNA phage with attractive characteristics for gene delivery, including a theoretically unlim...
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BMC
2023-07-01
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| Series: | Microbial Cell Factories |
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| Online Access: | https://doi.org/10.1186/s12934-023-02135-w |
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| author | Shirley Wong Salma Jimenez Roderick A. Slavcev |
| author_facet | Shirley Wong Salma Jimenez Roderick A. Slavcev |
| author_sort | Shirley Wong |
| collection | DOAJ |
| description | Abstract Background As simplistic proteinaceous carriers of genetic material, phages offer great potential as targeted vectors for mammalian transgene delivery. The filamentous phage M13 is a single-stranded DNA phage with attractive characteristics for gene delivery, including a theoretically unlimited DNA carrying capacity, amenability to tropism modification via phage display, and a well-characterized genome that is easy to genetically modify. The bacterial backbone in gene transfer plasmids consists of elements only necessary for amplification in prokaryotes, and, as such, are superfluous in the mammalian cell. These problematic elements include antibiotic resistance genes, which can disseminate antibiotic resistance, and CpG motifs, which are inflammatory in animals and can lead to transgene silencing. Results Here, we examined how M13-based phagemids could be improved for transgene delivery by removing the bacterial backbone. A transgene cassette was flanked by isolated initiation and termination elements from the phage origin of replication. Phage proteins provided in trans by a helper would replicate only the cassette, without any bacterial backbone. The rescue efficiency of “miniphagemids” from these split origins was equal to, if not greater than, isogenic “full phagemids” arising from intact origins. The type of cassette encoded by the miniphagemid as well as the choice of host strain constrained the efficiency of phagemid rescue. Conclusions The use of two separated domains of the f1 ori improves upon a single wildtype origin while still resulting in high titres of miniphagemid gene transfer vectors. Highly pure lysates of miniaturized phagemids could be rapidly obtained in a straightforward procedure without additional downstream processing. |
| format | Article |
| id | doaj-art-7f4cbd3fe97e40339f91a75a9be234f9 |
| institution | OA Journals |
| issn | 1475-2859 |
| language | English |
| publishDate | 2023-07-01 |
| publisher | BMC |
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| series | Microbial Cell Factories |
| spelling | doaj-art-7f4cbd3fe97e40339f91a75a9be234f92025-08-20T02:25:17ZengBMCMicrobial Cell Factories1475-28592023-07-0122111410.1186/s12934-023-02135-wConstruction and characterization of a novel miniaturized filamentous phagemid for targeted mammalian gene transferShirley Wong0Salma Jimenez1Roderick A. Slavcev2School of Pharmacy, University of WaterlooSchool of Pharmacy, University of WaterlooSchool of Pharmacy, University of WaterlooAbstract Background As simplistic proteinaceous carriers of genetic material, phages offer great potential as targeted vectors for mammalian transgene delivery. The filamentous phage M13 is a single-stranded DNA phage with attractive characteristics for gene delivery, including a theoretically unlimited DNA carrying capacity, amenability to tropism modification via phage display, and a well-characterized genome that is easy to genetically modify. The bacterial backbone in gene transfer plasmids consists of elements only necessary for amplification in prokaryotes, and, as such, are superfluous in the mammalian cell. These problematic elements include antibiotic resistance genes, which can disseminate antibiotic resistance, and CpG motifs, which are inflammatory in animals and can lead to transgene silencing. Results Here, we examined how M13-based phagemids could be improved for transgene delivery by removing the bacterial backbone. A transgene cassette was flanked by isolated initiation and termination elements from the phage origin of replication. Phage proteins provided in trans by a helper would replicate only the cassette, without any bacterial backbone. The rescue efficiency of “miniphagemids” from these split origins was equal to, if not greater than, isogenic “full phagemids” arising from intact origins. The type of cassette encoded by the miniphagemid as well as the choice of host strain constrained the efficiency of phagemid rescue. Conclusions The use of two separated domains of the f1 ori improves upon a single wildtype origin while still resulting in high titres of miniphagemid gene transfer vectors. Highly pure lysates of miniaturized phagemids could be rapidly obtained in a straightforward procedure without additional downstream processing.https://doi.org/10.1186/s12934-023-02135-wFilamentous bacteriophage M13PhagemidGene transferNon-viral gene deliveryDNA minivector |
| spellingShingle | Shirley Wong Salma Jimenez Roderick A. Slavcev Construction and characterization of a novel miniaturized filamentous phagemid for targeted mammalian gene transfer Microbial Cell Factories Filamentous bacteriophage M13 Phagemid Gene transfer Non-viral gene delivery DNA minivector |
| title | Construction and characterization of a novel miniaturized filamentous phagemid for targeted mammalian gene transfer |
| title_full | Construction and characterization of a novel miniaturized filamentous phagemid for targeted mammalian gene transfer |
| title_fullStr | Construction and characterization of a novel miniaturized filamentous phagemid for targeted mammalian gene transfer |
| title_full_unstemmed | Construction and characterization of a novel miniaturized filamentous phagemid for targeted mammalian gene transfer |
| title_short | Construction and characterization of a novel miniaturized filamentous phagemid for targeted mammalian gene transfer |
| title_sort | construction and characterization of a novel miniaturized filamentous phagemid for targeted mammalian gene transfer |
| topic | Filamentous bacteriophage M13 Phagemid Gene transfer Non-viral gene delivery DNA minivector |
| url | https://doi.org/10.1186/s12934-023-02135-w |
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