The Drosophila <i>attP40</i> docking site and derivatives are insertion mutations of <i>MSP300</i>

Graaf, Kevin van der; Srivastav, Saurabh; Singh, Pratibha; McNew, James A.; Stern, Michael

doi:10.1101/2022.05.14.491875

Cited by 6 publications

(10 citation statements)

References 37 publications

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“…As in trans- Tango (Talay et al, 2017), the panneuronal components are inserted at the attP40 docking site in the genome. It is noteworthy that the attP40 docking site that has recently been shown to cause problems in the nervous system, especially when homozygous (Duan et al, 2022; Groen et al, 2022; van der Graaf et al, 2022). Therefore, we advise against using the panneuronal components in a homozygous configuration.…”

Section: Discussionmentioning

confidence: 99%

retro-Tango enables versatile retrograde circuit tracing inDrosophila

Sorkaç

Moșneanu

Crown

et al. 2022

Preprint

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Transsynaptic tracing methods are crucial tools in studying neural circuits. Although a couple of anterograde tracing methods and a targeted retrograde tool have been developed inDrosophila melanogaster, there is still need for an unbiased, user-friendly, and flexible retrograde tracing system. Here we describeretro-Tango, a method for transsynaptic, retrograde circuit tracing and manipulation inDrosophila. In this genetically encoded system, a ligand-receptor interaction at the synapse triggers an intracellular signaling cascade that results in reporter gene expression in presynaptic neurons. Importantly, panneuronal expression of the elements of the cascade renders this method versatile, enabling its use not only to test hypotheses but also to generate them. We validateretro-Tango in various circuits and benchmark it by comparing our findings with the electron microscopy reconstruction of theDrosophilahemibrain. Our experiments establishretro-Tango as a key method for circuit tracing in neuroscience research.

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Section: Discussionmentioning

confidence: 99%

retro-Tango enables versatile retrograde circuit tracing inDrosophila

Sorkaç

Moșneanu

Crown

et al. 2022

Preprint

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“…Recently, a large portion of the RNAi lines from the VDRC KK collection were found to produce phenotypes due to a secondary insertion and consequent ectopic expression of the tiptop gene (Green et al , 2014; Vissers et al , 2016). More recently, van der Graaf and colleagues reported that transgenes inserted in the commonly used attP40 landing site on the second chromosome can decrease the expression of Msp300, a member of the LINC complex, and hence lead to nuclear size, shape and positioning phenotypes (van der Graaf et al , 2022).…”

Section: Discussionmentioning

confidence: 99%

DrosophilapVALIUM10 TRiP RNAi lines cause undesired silencing of Gateway-based transgenes

Stanković

Csordás

Uhlířová

2022

Preprint

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SUMMARYPost-transcriptional gene silencing using double-stranded RNA has revolutionized the field of functional genetics, allowing fast and easy disruption of gene function in various organisms. In Drosophila, many transgenic RNAi lines have been generated in large-scale efforts, including the Drosophila Transgenic RNAi Project (TRiP), to facilitate in vivo knockdown of virtually any Drosophila gene with spatial and temporal resolution. The available transgenic RNAi lines represent a fundamental resource for the fly community, providing an unprecedented opportunity to address a vast range of biological questions relevant to basic and biomedical research fields. However, caution should be applied regarding the efficiency and specificity of the RNAi approach. Here, we demonstrate that pVALIUM10-based RNAi lines, representing ∼11% of the total TRiP collection, cause unintended silencing of transgenes expressed from Gateway destination vectors generated via site-specific recombination. The silencing is mediated by targeting attB1 and attB2 sequences generated in the recombination reaction and included in the transcribed mRNA. Deleting these attB sites from the Gateway expression vector prevents silencing and restores expected transgene expression.

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“…A large portion of the RNAi lines from the VDRC KK collection were found to produce phenotypes because of a secondary insertion and consequent ectopic expression of the tiptop gene ( Green et al, 2014 ; Vissers et al, 2016 ). More recently, van der Graaf and colleagues reported that transgenes inserted in the commonly used attP40 landing site on the second chromosome can decrease the expression of Msp300, a member of the LINC complex, and hence lead to nuclear size, shape, and positioning phenotypes ( van der Graaf et al, 2022 Preprint ).…”

Section: Discussionmentioning

confidence: 99%

DrosophilapVALIUM10 TRiP RNAi lines cause undesired silencing of Gateway-based transgenes

2022

View full text Add to dashboard Cite

Post-transcriptional gene silencing using double-stranded RNA has revolutionized the field of functional genetics, allowing fast and easy disruption of gene function in various organisms. InDrosophila, many transgenic RNAi lines have been generated in large-scale efforts, including theDrosophilaTransgenic RNAi Project (TRiP), to facilitate in vivo knockdown of virtually anyDrosophilagene with spatial and temporal resolution. The available transgenic RNAi lines represent a fundamental resource for the fly community, providing an unprecedented opportunity to address a vast range of biological questions relevant to basic and biomedical research fields. However, caution should be applied regarding the efficiency and specificity of the RNAi approach. Here, we demonstrate that pVALIUM10-based RNAi lines, representing ∼13% of the total TRiP collection (1,808 of 13,410 pVALIUM TRiP–based RNAi lines), cause unintended off-target silencing of transgenes expressed from Gateway destination vectors. The silencing is mediated by targeting attB1 and attB2 sequences generated via site-specific recombination and included in the transcribed mRNA. Deleting these attB sites from the Gateway expression vector prevents silencing and restores expected transgene expression.

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The Drosophila attP40 docking site and derivatives are insertion mutations of MSP300

Cited by 6 publications

References 37 publications

retro-Tango enables versatile retrograde circuit tracing inDrosophila

retro-Tango enables versatile retrograde circuit tracing inDrosophila

DrosophilapVALIUM10 TRiP RNAi lines cause undesired silencing of Gateway-based transgenes

DrosophilapVALIUM10 TRiP RNAi lines cause undesired silencing of Gateway-based transgenes

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