phiC31-Integrase-Mediated Site-Directed Transgene Integration: A Microinjection Technique for Site-Specific Transgene Integration into an Anopheles Vector
Published: April 30, 2023
Abstract
Source: Adolfi, A., Lynd, A., Lycett, G. J., James, A. A. Site-Directed φC31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria. J. Vis. Exp. (2021).
In this video, we demonstrate phiC31-mediated site-specific transgene integration in the Anopheles embryo via microinjection. This method helps introduce desired genes into a host system for research and industrial applications.
Protocol
1. Preparation of plasmids for the microinjection mix NOTE: The protocol illustrated here involves the use of two plasmids: an attB (attachment site derived from the host bacterium)-tagged donor plasmid carrying the transgene of interest, and a helper plasmid that expresses the φC31 integrase under the regulation of the Drosophila Hsp70 promoter. Purify donor and helper plasmids using an endo…
Offenlegungen
The authors have nothing to disclose.
Materials
| 1.5 mL eppendorf tubes | |||
| EndoFree Plasmid Maxi Kit (10) | Qiagen | 12362 | |
| Ethanol, Absolute, Molecular Biology Grade | |||
| Halocarbon oil 27 | Sigma | H8773 | |
| Halocarbon oil 700 | Sigma | H8898 | |
| Petri dishes | |||
| Potassium chloride | |||
| Sodium Chloride | |||
| Sodium phosphate dibasic | |||
| Sodium Acetate Solution (3 M), pH 5.2 | Thermo Fisher Scientific (Life Technologies) | R1181 | |
| Stable brush Size 0 |
Tags
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phiC31-Integrase-Mediated Site-Directed Transgene Integration: A Microinjection Technique for Site-Specific Transgene Integration into an Anopheles Vector. J. Vis. Exp. (Pending Publication), e20974, doi: (2023).