Embryo Microinjection for Transgenesis in Drosophila

Summary

This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and presents an optimized protocol for embryo microinjection, a crucial step for creating transgenic flies.

Abstract

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the construction of transgenic flies. Microinjection requires some types of equipment, including a microinjector, a micromanipulator, an inverted microscope, and a stereo microscope. Plasmids isolated with a plasmid miniprep kit are qualified for microinjection. Embryos at the pre-blastoderm or syncytial blastoderm stage, where nuclei share a common cytoplasm, are subjected to microinjection. A cell strainer eases the process of dechorionating embryos. The optimal time for dechorionation and desiccation of embryos needs to be determined experimentally. To increase the efficiency of embryo microinjection, needles prepared by a puller need to be beveled by a needle grinder. In the process of grinding needles, we utilize a foot air pump with a pressure gauge to avoid the capillary effect of the needle tip. We routinely inject 120-140 embryos for each plasmid and obtain at least one transgenic line for around 85% of plasmids. This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and presents a detailed protocol for embryo microinjection for transgenesis in Drosophila.

Introduction

The fruit fly Drosophila melanogaster is extremely amenable to genetic manipulation and genetic analysis. Transgenic fruit flies are widely used in biological research. Since it was developed in the early 1980s, P-element transposon-mediated transgenesis has been indispensable for Drosophila research¹. In some scenarios, other transposons, such as piggyBac and Minos have been used for transgenesis in Drosophila as well². Transgenes via transposon are randomly inserted into the Drosophila genome, and the expression levels of transgenes at different genomic loci may vary due to position ....

Protocol

1. Preparation of plasmids

1. Isolate plasmids from 4 mL overnight bacterial cultures using a plasmid miniprep kit. Elute with 40 µL of elution buffer.
   NOTE: Isolation of plasmids using a plasmid midi-prep kit is not necessary. A plasmid miniprep kit can fully meet the experimental requirements for embryo microinjection. In this protocol, the prepared UAS-cDNA/ORF plasmids contain ten copies of UAS, an Hsp70 minimal promoter, an attB site, a mini-white gene, and a gene of interest.

Discussion

Here, we present a protocol for embryo microinjection for transgenesis in Drosophila. For phiC31-mediated site-specific transgenesis, we injected 120-140 embryos for each plasmid and obtained at least one transgenic line for around 85% of plasmids (Supplementary Table 2). In our experience, plasmid DNA isolated by a plasmid miniprep kit suffices for transgenesis in Drosophila. Plasmid DNA concentrations ranging from 20 ng/µL to 1683 ng/µL might have no apparent effect on the s.......

Materials

Name	Company	Catalog Number	Comments
100 μm Cell Strainer	NEST	258367
3M double-sided adhesive	3M	415
Borosilicate glass	SUTTER	B100-75-10
Diamond abrasive plate	SUTTER	104E
FLAMING/BROWN Micropipette Puller	SUTTER	P-1000
Foot air pump with pressure gauge	Shenfeng	SF8705D
Halocarbon oil 27	Sigma	H8773
Halocarbon oil 700	Sigma	H8898
Inverted microscope	Nikon	ECLIPSE Ts2R
Microinjection pump	eppendorf	FemtoJet 4i
Micromanipulator	eppendorf	TransferMan 4r
Micropipette Beveler	SUTTER	BV-10
Microscope cover glasses (18 mm x 18 mm)	CITOLAS	10211818C
Microscope slides (25 mm x 75 mm)	CITOLAS	188105W
Petri dish (90 mm x 15 mm)	LAIBOER	4190152
Photo-Flo 200	Kodak	1026269
QIAprep Spin Miniprep Kit	Qiagen	27106
Stereo Microscope	Nikon	SMZ745