phiC31-Integrase-Mediated Site-Directed Transgene Integration: A Microinjection Technique for Site-Specific Transgene Integration into an Anopheles Vector

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.

1. Purify donor and helper plasmids using an endotoxin-free plasmid purification kit.
   NOTE: Sequence the final plasmid preparation used for injection to verify the integrity o....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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