Eppendorf Microinjection of cDNA into Drosophila embryos User Manual
Userguide
No 041 I Micromanipulation
Microinjection of cDNA into
Enrica Charbonnier, Devolopmental Biology, Biology I, University of Freiburg, 79104 Freiburg, Germany,
enrica.charbonnier@googlemail.com
Abstract
Germline transformation via microinjection of DNA into Drosophila melanogaster has been used to interfere with
gene expression and study their function for many years. Recently, Bischof and co-workers established a new system to circumvent typical limitations by optimizing a phiC31-based integration system [1]. This integration system
demonstrates to be extremely efficient in Drosophila embryo microinjection. Furthermore, in combination with the
ease-of-use plus versatility of this technique, a systematic high throughput screening of large cDNA sets and regulatory elements is now feasible.
In this Userguide, a step-by-step protocol using the Eppendorf programmable microinjector FemtoJet and ready to
use Femtotip II microcapillaries is presented for this application.
Introduction
Nowadays the scientific community invests great effort into
the identification and characterization of all genes which
are relevant to a specific signalling pathway or biological
process and their interaction between each other. Since
long the Drosophila genome has been sequenced and thus
makes it a powerful tool to study gene expression and gene
regulation even at the transcriptional level. P-elements are
a powerful tool to generate insertion mutagenesis thanks to
their random integration into the Drosophila genome. However, this random insertion causes difficulties to analyze the
expression pattern of the transgenic animal obtained with
this method. One of the biggest problems is the positional
effect, because the genomic environment can influence the
expression of the transgene.
Recently, the genome integration method based on the
site-specific phiC31 integrase [2], has been applied to
Drosophila [3].
Drosophila
The bacteriophage phiC31 encodes a serine integrase
that mediates sequence-directed recombination between
a bacterial attachment site (attB) and a phage attachment
site (attP) [4]. Bischof and co-workers generated a collection of Drosophila lines (available now at Bloomington
Drosophila Stock Center, http://flystocks.bio.indiana.edu/)
with precisely mapped attP sites that allow the insertion of
transgenes into many different predetermined intergenetic
locations throughout the fly genome. By using regulatory
elements of the nanos and vasa genes, they established
endogenous sources of the phiC31 integrase, eliminating
the difficulties of coinjecting integrase mRNA and raising
the transformation efficiency. Moreover, to discriminate
between specific and rare non-specific integration events, a
white gene-based reconstitution system was generated that
enables visual selection for precise attP targeting [1].
embryos
Userguide No 041 | Page 2
Fig. 1: Workstation for Drosophila injection, consisting of a Leica DM IL inverted microscope, a Leica manual micromanipulator and
an Eppendorf programmable microinjector FemtoJet.
Materials and Methods
DNA is introduced at 18 °C in Drosophila embryos by
microinjection, into the posterior end of the egg just before
pole cell formation using a manual micromanipulator (Leica
microsystems), Eppendorf programmable microinjector, the
FemtoJet, and ready to use, sterile Femtotip II microinjection
capillaries (see figure 1).
Instruments
‡ Inverted microscope (Leica DM IL, Leica Microsystems
CMS GmbH, Wetzlar, Germany)
‡ Manual Micromanipulator (Leica microsystems)
Programmable microinjector FemtoJet (Eppendorf AG,
Hamburg, Germany)
‡ Micropipette 0.5-10 µL
‡ Microcentrifuge Eppendorf 5424
Hamburg, Germany)
(Eppendorf AG,
Materials
‡
Femtotip II microinjection capillaries (Eppendorf AG,
Hamburg, Germany)
‡
Microloader (Eppendorf AG, Hamburg, Germany)
‡
Microscope slide 76x26 mm (Leica Microsystems)
Agar plates with grape juice
‡
Small mesh basket for collecting rinsing and dechorionat ing embryos
‡
Double sided tape (tesa SE, Hamburg, Germany)
‡
Embryo collection chambers
‡
Sodium hypochlorite (Sigma-Aldrich, Germany)
‡
Hair dryer
‡
Voltalef oil (VWR, Bruchsal, Germany)
‡
Plasmid purification kit (Qiagen, Hilden, Germany)
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