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…

Divulgations

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

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Citer Cet Article
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).

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