Academic Journal
Inducible Protein Knockdown via Engineered Small RNAs in Chlamydia.
| Τίτλος: | Inducible Protein Knockdown via Engineered Small RNAs in Chlamydia. |
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| Συγγραφείς: | Densi A; Department of Developmental and Cell Biology, University of California, Irvine, CA, USA., Tan M; Department of Microbiology and Molecular Genetics and Department of Medicine, University of California, Irvine, CA, USA., Sütterlin C; Department of Developmental and Cell Biology, University of California, Irvine, CA, USA. suetterc@uci.edu. |
| Πηγή: | Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2027; Vol. 3073, pp. 231-241. |
| Τύπος έκδοσης: | Journal Article |
| Γλώσσα: | English |
| Στοιχεία περιοδικού: | Publisher: Humana Press Country of Publication: United States NLM ID: 9214969 Publication Model: Print Cited Medium: Internet ISSN: 1940-6029 (Electronic) Linking ISSN: 10643745 NLM ISO Abbreviation: Methods Mol Biol Subsets: MEDLINE |
| Imprint Name(s): | Publication: Totowa, NJ : Humana Press Original Publication: Clifton, N.J. : Humana Press |
| Ιατρικοί όροι (MeSH): | Gene Knockdown Techniques*/methods , Chlamydia trachomatis*/genetics , Bacterial Proteins*/genetics , RNA, Small Untranslated*/genetics, Genetic Engineering/methods ; RNA, Bacterial/genetics ; RNA, Small Interfering/genetics ; 5' Untranslated Regions ; Humans |
| Περίληψη: | Genetic manipulation of Chlamydia trachomatis is challenging because of its obligate intracellular lifestyle and reduced genome with a high proportion of essential genes. Functional analysis of chlamydial genes often requires alternative genetic strategies such as conditional knockdown rather than gene disruption. This chapter describes the use of an engineered small RNA (sRNA) to achieve inducible protein knockdown in C. trachomatis. This approach utilizes the C. trachomatis sRNA CtrR3 as a scaffold to express a targeting sequence that interferes with a target mRNA by binding its 5' untranslated region (UTR), in the region of the ribosome-binding site (RBS) and start codon. The protocol detailed here covers design of the targeting sequence, bioinformatic analysis to minimize off-target binding, construction of the knockdown sRNA plasmid, generation of a chlamydial transformant expressing the engineered sRNA, induction of protein depletion, assessment of knockdown efficiency and phenotypes, and complementation to demonstrate knockdown specificity. (© 2027. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.) |
| Competing Interests: | Conflict of Interest: The authors have no conflicts of interest to declare that are relevant to the content of this chapter. |
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| Contributed Indexing: | Keywords: Chlamydia; Complementation; Engineered sRNA; Knockdown specificity; Off-target effects; Post-transcriptional repression; Protein depletion; Protein knockdown |
| Substance Nomenclature: | 0 (Bacterial Proteins) 0 (RNA, Small Untranslated) 0 (5' Untranslated Regions) 0 (RNA, Bacterial) 0 (RNA, Small Interfering) |
| Entry Date(s): | Date Created: 20260922 Date Completed: 20260922 Latest Revision: 20260922 |
| Update Code: | 20260923 |
| DOI: | 10.1007/978-1-0716-5531-3_17 |
| PMID: | 42771154 |
| Βάση Δεδομένων: | MEDLINE |
| ISSN: | 1940-6029 |
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| DOI: | 10.1007/978-1-0716-5531-3_17 |