| Autor: |
Natsuki Matsushita, Shigeki Kato, Kayo Nishizawa, Masateru Sugawara, Kosei Takeuchi, Yoshiki Miyasaka, Tomoji Mashimo, Kazuto Kobayashi |
| Jazyk: |
angličtina |
| Rok vydání: |
2023 |
| Předmět: |
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| Zdroj: |
Cell Reports: Methods, Vol 3, Iss 2, Pp 100393- (2023) |
| Druh dokumentu: |
article |
| ISSN: |
2667-2375 |
| DOI: |
10.1016/j.crmeth.2022.100393 |
| Popis: |
Summary: The flip-excision switch (FLEX) system with an adeno-associated viral (AAV) vector allows expression of transgenes in specific cell populations having Cre recombinase. A significant issue with this system is non-specific expression of transgenes in tissues after vector injection. We show here that Cre-independent recombination events in the AAV genome carrying the FLEX sequence occur mainly during the production of viral vectors in packaging cells, which results in transgene expression in off-target populations. Introduction of a relatively longer nucleotide sequence between two recognition sites at the unilateral side of the transgene cassette, termed a unilateral spacer sequence (USS), is useful to suppress the recombination in the viral genome, leading to the protection of non-specific transgene expression with enhanced gene expression selectivity. Our FLEX/USS system offers a powerful strategy for highly specific Cre-dependent transgene expression, aiming at various applications for structural and functional analyses of target cell populations. Motivation: There is evidence for non-specific expression of transgenes in off-target cell populations after injection of AAV vectors carrying the FLEX system into tissues, but understanding of the molecular mechanism that produces this non-specific transgene expression has been limited. Here, we investigated the possibility that Cre-independent recombination events are predominantly generated in viral genomes with the FLEX sequence during vector production in cultured cells. We then aimed to develop a viral vector technology with the USS to protect non-specific transgene expression, enhancing the selectivity of Cre-dependent gene expression in target cell populations. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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