Integrating scFv-mNeonGreen-GB1 into Drosophila

The scFv sequence was obtained from plasmid pHR-scFv-GCN4-sfGFP-GB1-NLS-dWPRE (Addgene #60906) (Tanenbaum et al., 2014 (link)). scFv-mNeonGreen-GB1-NLS was assembled using In-Fusion HD Cloning Plus multiple insert cloning (Takara Bio #638911) and integrated into the KpnI/SpeI sites of pCasper-attB-nos>tub3′UTR+1 kb (containing the nos promoter in EcoRI to direct maternal expression and tubulin 3′UTR+1 kb of downstream sequence in XbaI). To generate the noNLS version, the scFv-mNeonGreen-GB1 was amplified by PCR and inserted into KpnI/NotI of pCasper-attB-nos>tub3′UTR. ϕC31-based integration was used for specific re-integration of the transgene into sites 25C6 (chr2) and 86Fb (chr3) by the University of Cambridge microinjection service.

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Vinter D.J., Hoppe C., Minchington T.G., Sutcliffe C, & Ashe H.L. (2021). Dynamics of hunchback translation in real-time and at single-mRNA resolution in the Drosophila embryo. Development (Cambridge, England), 148(18), dev196121.

Publication 2021

In-Fusion HD Cloning Plus

Ecori Maternal Microinjection Plasmid Transgene Tubulin

Corresponding Organization :

Other organizations : University of Manchester

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Variable analysis

independent variables

Presence or absence of the nuclear localization signal (NLS) in the scFv-mNeonGreen-GB1 construct

dependent variables

Localization of the scFv-mNeonGreen-GB1 construct (with or without NLS)

control variables

Plasmid constructs used (pHR-scFv-GCN4-sfGFP-GB1-NLS-dWPRE and pCasper-attB-nos>tub3′UTR+1 kb)
Cloning method (In-Fusion HD Cloning Plus)
Integration site (25C6 on chr2 and 86Fb on chr3)
Expression system (nos promoter and tubulin 3′UTR+1 kb)

controls

Positive control: scFv-mNeonGreen-GB1-NLS construct
Negative control: scFv-mNeonGreen-GB1 construct without NLS

Annotations

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