Biomedical Genomics, Master's Programme

Developing electroporation as a method to obtain Stable

Transformation in Drosophila melanogaster

Fuad Ali Akbar Ali

Supervisor: Docent Per Kylsten

Examiner: Professor Einar Hallberg

October, 2008 Södertörn Högskola (Södertörn University College)

Developing electroporation as a method to obtain Stable
Transformation in Drosophila melanogaster

Abstract

In this project I have tried to obtain stable transformants of Drosophila

melanogaster flies using electroporation. I have completed
approximately 200 tests using different DNA concentrations, voltages
and cuvettes, including a novel Petri dish cuvette which I developed and
manufactured myself. I also developed new and more efficient
procedures of egg collection and egg dechorionation. Although I was not
successful in obtaining true stable transformants, control experiments
indicate that electroporation of DNA into embryos could be
accomplished under the conditions used. The lack of stable
transformants was probably due to failure of the electroporated DNA to
integrate into the host genome. The reasons for why the DNA did not
integrate was not further investigated in this study.
_____________________________________________________________

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Introduction

Transposable elements
In order to obtain stable transformants of Drosophila melanogaster P -
elements which work in the germline cells are often used. P-elements are
transposable elements that have the ability to move within their host genome
from one genomic place to another. Because of these properties, P-elements
have been an attractive tool for researchers in the field of biological sciences
studying the molecular mechanism and control factors of these elements.
The use of P-elements for transgenesis was first developed by Rubin and
Spradling (1), who restored wild type function to rosy mutant flies by
injecting a P-element containing a functional rosy gene into Drosophila
embryos and recovering rescued flies among the progeny of the injected
individuals (2). Since then, P-elements have been widely adapted and
modified to provide a range of functional tools for biologists; they can be
used for gene tagging, gene disruption, chromosome engineering and
inducible gene expression.

Construction of P-element
P-elements are believed to have entered the Drosophila melanogaster
population nearly 100 years ago by horizontal transfer from another
Drosophila species and since then have spread to most wild and laboratory
populations. P-elements can be classified into two types, autonomous
elements, which encode their own source of the transposase to be able to
move, and secondly non-autonomous elements that need an external source
of transposase for mobilisation.
The wild-type autonomous P element is 2.9 kb in size and contains a four-
exon transposase gene and a number of inverted repeats. Sequence analysis
of several P-elements reveal that not more than 138 bp at the 5’end and 216

3
bp at the 3’end are necessary for transposition or excision (3) and in order to
transpose, all P-elements must have intact 31 pb perfect inverse terminal
repeats and 11 bp subterminal inverted repeats, the repeats are the site for the
activity of transposase. P-element transposition is in a natural way restricted
to germline cells because the splicing of the intron between exons 2 and 3 of
the transposase gene is inhibited in somatic cells. This is due to a splicing
repressor protein (4) (5). In the soma, the splicing of the three remaining
exons in somatic cells leads to the production of a truncated transposase
protein that acts as a repressor of P-element mobility(6). This truncated
repressor is also responsible for the fact that, in wild type strains, P elements
mobility is restricted to crosses between M strain females and P strain males,
since P strain females pass on the repressor protein through the cytoplasm of
their eggs(7)(8). Once this was understood, it was relatively easy to engineer
the transposase gene, by deleting the regulated intron, to produce a
transposase source, ∆2-3, that will function in any tissue of Drosophila
melanogaster fly (9).

Transformation
Transformation of transgenic P- elements requires either co-injecting a
transposable construct with an element that produces transposase or
introducing a construct into an embryo that carries an autosomal copy of the
∆2-3 transposase source (10) (11). The P-elements has become the basic tool
in generating genomic transfomants of Drosophila melanogaster fly.

The Electroporation Method

The process of transformation in fly embryos through direct micro-injection
is relatively difficult. It is tough on embryos, requires high skill and long
periods of time. For the purpose of development of the transformation and
shortening the time to obtain results and better performance a new
technology, the Electroporation Method, has been used. This technique uses

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an Electroporator. This electric device provides variable voltage and
capacitor values and electrodes by which electric pulses pass throughout the
solution containing embryos and the DNA. These impulses perforate the
cells and facilitate the permeation of DNA to the inside of the cells. Research
has shown that various factors play a role in this method, like biological
variations, membrane structure and its components, and the sensitivity of
each cell and it's sustainability to electrical pulses. The cell-to-cell biological
variability causes some cells to be more sensitive to electroporation than
others. The plasma membrane of a cell is largely composed of two layers of
molecules (phospholipid bilayer) where the polar hydrophilic head groups
face outward, and the non-polar hydrophobic tail groups face inward and
interact with one another to hold the membrane together (figure-1). Thus the
cell can protect itself from the external environment and any

Figure-1 Hypothesized way of the electroporation

polar molecules, including DNA and protein, cannot pass through this
membrane without relatively strong external force (12) (13).
The number of pores and effective pore radius increase proportional to the
product of the "amplitude" and "duration" of an electric pulse. For
electropores to be induced, the product of the pulse amplitude and the pulse

5
duration has to be above a lower limit threshold. This threshold is largely
dependent on the reciprocal of the cell size. If the upper limit threshold is
reached pore radius and total pore area are too large for the cell to be
repaired by any spontaneous or biological process. The result is irreversible
damage to the cell or cell lyses. Because the mechanism of electroporation is
not well understood, further experiments will be needed to fully understand
how this method works in detail (14) (15).

Previous studies
Note that previous studies have proven the possibility of using this technique
successfully. Finnerty et al. (16) demonstrated that it is possible to transform
Drosophila melanogaster embryos by electroporation. Finnerty et al. have
introduced into mutant Drosophila melanogaster embryos both plasmid
DNA carrying the gene for aldehyde oxidase (AO) and phage DNA carrying
the maroon-like (ma-l) gene that provides a sulphured form of molybdenum
cofactor required for aldehyde oxidase activity. Plasmid DNA from crude
boiling preparations (17) and phage DNA was prepared by sedimentation in
CsCl (18) at a concentration of 10 µg/mL in electroporation buffer with
5mM KCl, 0.1mM sodium phosphate buffer, pH 7.8(19). They used the Bio-
Rad (Richmond, CA) Gene Pulser, model 165-2076 with Capacitance
Extender model 165-2087. Cuvettes used were: catalog number 165-2086,
2mm electrode gap (Bio-Rad). Dechorionation solution used was common
household bleach (Clorox) diluted to 50% with distilled water. Egg-laying
chambers were made from disposable 50 mL polypropylene tubes with plug
seal cap (catalog number 05-539-6, Fisher Scientific, Springfield, NJ). They
cut the lower conical part of the tube off (at the 40 mL mark) and tightly
covered the threaded end of the tube with Nitex nylon mesh (500 µm pore
size). Egg-collection basket used was also made from the same 50 mL tubes.
They cut a 1.5 cm² window in the center of the screw cap and covered the

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threaded tube with a 7 cm² piece of Nitex nylon mesh (100 µm pore size)
and held the mesh in place with the screw cap. They used both
dechorionated and non-dechorionated embryos in their experiments.
However, the experiments performed by Finnerty et al. (16) were only
designed to generate transient transformants. The aim of this project is to
develop an electroporation-based method for simple and effective generation
of stable genomic Drosophila melanogaster transformants.

Materials and Methods

Fly stocks
The recipient stock of Drosophila melanogaster has a stable source of
transposase ( w[*]; wg[Sp-1]/CyO; ry[506] Sb[1] P{ry[+t7.2]=Delta2-
3}99B/TM6B, Tb[+] ). It was obtained from the Bloomington center (IN,
USA ) stock # 3629 and called ∆2-3 flies in this report. The flies were used
to generate red eyed stably transformed flies.
For the purpose of testing electroporation or the direct micro-injection
efficacy I have used these GAL4 drivers strains: stock # 4442, ( y[1] w[*];
P{w[+mC]=GAL4-nos.NGT}40 ), stock # 1973, ( y[1] w[*];
P{w[+mW.hs]=en2.4-GAL4}e22c/SM5 ) and stock # 5460, ( w[*];
P{w[+mW.hs]=GAL4-da.G32}UH1 ), which were obtained from the
Bloomington center(IN, USA). Embryos were assayed by ZEISS uv-
microscope model, Stemi SV11 APO with light resource ebq100.

Plasmid vectors
pUAST and pUASP are vectors for constructing a UAS responder of the
gene of interest(20). In this project I have used the P-element vector,
p{UAST} prepared by QIAfilter Plasmid Midi Kit-25 Cat. NO. 12243(figure
2) for the ∆2-3 flies

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Figure 2 map of pUAST vector

and plasmid vector named pUASP-EGFP-C1-Nup35(a kind gift from Dr. M.

Rahman) prepared by QIAfilter Plasmid Midi Kit (25) Cat. NO.12243(figure
3) for the GAL4 flies.

Figure 3 map of pUASP vector

Electroporator: A BTX Electro Cell Manipulator 600 Electroporation

System (figure 4) was used. Its unit features are low voltage mode LOW
VM: electroporation pulse amplitude: 5 -
500 V Peak, capacitance Range: 25-3175
µF, high voltage mode HIGH VM:
electroporation pulse amplitude: 50-2500 V
Peak (capacitance is not used) and timing
resistors: from R1 to
R10,13,24,48,72,129,186, 246,360,480,720
ohms respectively. Figure-4 Electroporator BTX 600

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Cuvettes: I have used the same cuvettes as were used by Finnerty et al. The
cuvettes are catalog number 165-2086 with 1mm electrode gap and catalog #
165-2089 with 2mm electrode gap; (Bio-Rad cuvettes) (figure 5).
Egg lay agar: 20g food quality agar in 400 ml distilled water, 15g sugar 10
ml concentrated mixed fruit juice (Blandsaft), ½ tsp powdered carbon and 6
ml Nipagin (10% solution methyl-p-hydroxybenzoate in EtOH).

Figure-5 Bio-Rad Cuvettes

Electroporation buffer: 5mM KCl, 0.1mM sodium phosphate buffer, pH

7.8.
Dechorionation solution: common household bleach (Chlorine) diluted to
50% with distilled water.
Egg-collection basket made from 50 mL tube. A 1.5 cm² window was cut in

Figure-6 Egg-lay cage covered by Egg-lay cap

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the center of the screw cap. The threaded tube was covered with a 9 cm²
piece of Nitex nylon mesh (100 µm pore size) which held the mesh in place
with the screw cap. I transferred about 100 young Drosophila melanogaster
flies onto Egg-laying cage covered by an Egg-laying cap, a small Petri dish
containing egg laying agar with beads of dried yeast, sealed around its edge
by tape (figure 6). At the outset in any series of experiments involving
electroporation I discarded the first harvest to avoid older embryos. In the
subsequent harvest of embryos, Egg-laying period was 30 to 45 minutes at
room temperature (25˚C). Embryos were rinsed in sterilized water then
transferred by pipette into a tube.
After the disposing of excess water, buffer and DNA solution was added to
embryos which were then transferred into a cuvette with 1mm or 2mm
electrode gap for immediate electroporation of the non-dechorionated
embryos.
Embryo dechorionation was performed by immersing the egg-collection
basket containing washed embryos in 50% household bleach (chlorine)
(dechorionation solution) for 1 minute. After dechorionation, the embryos
were thoroughly rinsed in distilled water, and the excess water was disposed
by gently pressing a paper towel to the
outside of the mesh screen. Using a
0.3cm paintbrush the embryos were
immediately placed into a cuvette
containing buffer and DNA and
electroporated as described for non-
dechorionated embryos.

Figure-7
For the control tests we used GAL4- A and B are two pieces of double sided
Drosophila melanogaster flies. These sticky tape pasted on a glassy micro-slide.

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flies were used as a source for embryos to be used for direct micro-injection
and electroporation.
30-min egg-laying period embryos were used for electroporation or micro-
injection. Embryos were dechorionated by manual procedure using a blunt
needle and double sided sticky tape. I pasted two pieces of this tape, one 4cm
in length and 1mm width -A- and another 2 cm² area -B- on a micro-slide
(figure-7). I wetted both two pieces of tape by a small quantity of water and
transferred a number of the embryos on the -B- piece. After the
dechorionation I lined up the embryos on the -A- piece. For the
electroporating -A- piece was carefully removed and put into the Petri-dish
cuvette containing electroporation buffer but for the micro-injecting the -A-
piece was removed and pasted on another micro-slide then the embryos
covered with as little halocarbon oil mix (series HC-700 35ml + series 27
5ml) as possible.
The micro-injection set-up consists of an inverted microscope and an air-
pressure injecting device connected to a needle holder. The needles were
1.0mm OD borosilicate capillaries with omega dot fiber (e.g. Frederik Haer
& Co, # 30-30/0 and pulled on a horizontal micropipette puller of the Sutter
brand series (21).
For comparison of different tools and methods I separated a number of
embryos which were not electroporated, but otherwise treated in the same
way from the same samples and compared the rates of fly survival
electroporated relative.
Stable transformation was monitored by eye color where non-mosaic red
eyes would indicate stable transformation. This was monitored for both G0
(parental generation) and F1(first offspring generation).

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Results
I observed some problems during the initial experiments i.e. embryos would
break to a variable extent when transferring them into the electroporation
buffer leaking their contents, thus causing the electroporation conditions to
be variable. Also, some embryos stuck to the internal aspects of the Bio-Rad
cuvettes, outside of the buffer solution influencing the results.
New procedure
To avoid these problems I developed a new procedure. Instead of agar I used
a thick piece of black textile wetted with fruit juice, sprayed the yeast in the

Figure-8 The thick piece of black textile wetted Figure-9 Petri dish cuvette is designed to be
with fruit juice in the cap and the needle which we watched under the microscope.
used.

center of the piece of textile (figure-8). I found that flies put eggs in or on the
textile around the yeast without mixing with it. This is what enabled me to
complete the collection and use of embryos directly with a blunt needle,
without the need for rinsing, washing and using the pipette.
Development of Petri dish cuvette
I also manufactured two 1mm gap Petri dish cuvettes, each consisting of
two pieces of stainless steel functioning as electrodes, 4cm in length and
1mm or 2mm in width each linked to an electric wire installed properly on a
micro-slide inside of the Petri dish (figure 9).I lined up the embryos with

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the blunt needle, one at a time, on double sided sticky tape (SARSTEDT) to
ensure that all embryos would become subject to the charge.

A chart of the Ptri dish cuvette in Figure-9

The new Petri dish cuvette is designed to be monitored under the

microscope, thus I was able to observe the process accurately and see the
impact of the pulse on the solution and the embryos directly in the
microscope. It also enabled me to control the direction of pulses by replacing
the linking wires. Thus, I have been able to do two pulses with opposite
directions. Because the DNA is negatively charged, in the first pulse the
DNA will move toward one side of the cuvette and collect there. Another
counter pulse makes DNA introduction into the embryos possible better than
the first pulse.
The experiments
My work can be divided into three stages. Each stage determined by the
methods and tools which I used. Thus, the results obtained, shown in the
attached tables, are in accordance with these stages.

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In the first stage I tried to apply the procedure, tools and voltage values that
are mentioned in the Finnerty method taking into consideration some other
proposals listed by Ti-Fei Yuan (22).
Impact factors
The test results of the first stage set out in Tables 1, 2 and 3; indicated a
number of factors that could have had an impact on the embryos: some of
these factors are specific to the function of the electoporator system and
others are produced from the working methods and different tools. Key
variables in the system are three: the voltage, the capacitance and the
resistance. All these have central roles in the process of electroporation,
since any change in the values of these variables affects the embryos
significantly. The experiments have shown that the survival of the embryos
is affected mainly by the pulse duration which can be controlled by the
change in the
capacitance or the resistance, or both together.
In the first set of experiments, I used the non-
dechorionated embryos with 40 µl electroporation
buffer, DNA at concentration of 10 µg/ml using
different cuvettes. I found a significant decline in the
proportion of surviving embryos with higher
voltage.
Figure-10
Some of the embryos died direct without showing
Positive result using DNA at
any manifestations of growth. Others completed concentration of 1000 µg/ml by

development into larva but they died at different micro-injection using dechorionated
GAL4-embryos. A-arrow non-
instars. I have also noted that some larvae suffer
injectet embryo and B-arrow
from malformation in parts of the mouth and others injected embryo.

died because they have not been able to find the food.
Control tests without electroporation using the traditional way of egg-
collection and embryo preparation showed that embryo survival did not

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exceed 30%. This means that the ∆2-3 flies I used as recipients were already
weak.
I set up mating between the flies which survived electroporated but I did not
find any red-eyed flies in the parental of later generations.
In the second set of experiments I used non-dechorionated embryos with
different cuvettes, 40 µl electroporation buffer, different DNA
concentrations and various amounts of electroporation buffer while
continuing to use various cuvettes. The test results are set out in Tables 4 and
5.

At this stage I took into consideration the results obtained in the first set of
experiments and tried to focus on the values of the relatively moderate
voltages. As a result I got a good number of flies for subsequent mating but
there were no red-eyed flies in later generations and I
noted that some of the flies died in the pupa phase or
as young flies.
I changed the way of Egg-laying and Egg-collection
in this experimental set and did a number of control
tests without electroporation using ∆2-3 embryos. I
found the survival of embryos be 45% using this L R
method so it became apparent that the new Figure-11
procedure of embryo preparation is better than the Electroporated dechorionated
GAL4-embryos in (R) right side
traditional procedure.
and control embryos without
electroporation in the (L) left side.

Evidence of DNA introduction

In the third set of experiments I tried to obtain evidence of introducing DNA
into the embryos which was not possible to obtain in ∆2-3embryos because I
could not watch any manifestations of the introduction of the DNA in the
embryonic phases, I used GAL4 embryos as recipients to express the

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introduced DNA which encodes GFP in the early embryonic phases. Table 6,
summarises micro-injection experiments on dechorionated GAL4-embryos
using different DNA concentrations. I obtained a positive result, by help
from my supervisor Per Kylsten, using DNA at concentration of 1000 µg/ml
(figure10).

To test the possibility of DNA introduction by electroporation a similar

experiment was set up using dechorionated GAL4-embryos as recipients, in
Petri dish cuvette, 40 µl electroporation buffer and 1000 µg/ml DNA
concentrations, (Table 7), I obtained a positive result (figure-11) by using
two pulses with 350 v/cm at 50 µF in opposite directions, (Exp. 7-Table 7).
Expression levels of GFPs were less than what was attainable with the
microinjection method.
Control tests without electroporation using various stocks of GAL4 flies by
my new procedure of embryos preparation indicated an embryo survival rate
about of 60%. This means that the GAL4 flies are more vigorous than the
∆2-3 flies.
A series of experiments using dechorionated ∆2-3 embryos with Petri dish
cuvette, 40 µl electroporation buffer and 1000 µg/ml DNA concentrations
did not produce any surviving flies using the same values that were used for
the electroporation of GAL4-embryos. This is consistent with the ∆2-3 strain
being weaker. Electroporation using milder conditions did give flies, but
mating of these flies did not result in red-eyed progeny. The results are
shown in Table 8.

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Discussion

I have tried to clarify the problems associated with introducing DNA into fly
embryos using the electroporation method. I had a number of problems
during different stages of the work, which I think were the cause of the
failure to obtain stable transformant flies. The problems were that the ∆2-3
embryos were very weak, the electroporator was old and sometimes not
working well and I could not make control test for transgenic DNA-
integration into the genome by micro-injection into ∆2-3 embryos, because
of the long time it would take to obtain results.
I can conclude that if I had used another stock of flies with a greater
tolerance, maybe I could have obtained stable transformant flies. Further
testing, the use of more suitable modern electroporator and taking into
account my new procedure for embryo preparation and my Petri-dish
cuvettes may allow us to achieve our goal: to obtain stable transformation
using electroporation.

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Tables
Tables abbreviations:
V.; volt
T.C.; time constant
R.; resistance
C.; capacitor
E.E.; number of electroporated embryos
L.H.; number of larvae hatched
B.R.; Bio-Rad cuvette
P.d.; Petri dish cuvette
Table [1] Electroporation experiments using non-dechorionated embryos with Rad-Bio
cuvette 0,1cm with 40 µl electroporation buffer and DNA at concentration of 10 µg/ml.
Exp. Set V. Peak V. T. C. R. C. E.E. L.H.
1. 1500v/cm 1150v/cm 0,545 ms 13 Ohm not used 60 NO
2. 1250v/cm 960 v/cm 0,515 ms 13 Ohm not used 60 NO
3. 1250v/cm 990 v/cm 0,525 ms 13 Ohm not used 60 NO
4. 1250v/cm 920 v/cm 0,558 ms 13 Ohm not used 60
250 v/cm 170 v/cm 0,599 ms 13 Ohm not used 60 NO
5. 1000v/cm 700 v/cm 0,509 ms 13 Ohm not used 60 2
6. 1000v/cm 770 v/cm 0,535 ms 13 Ohm not used 60 NO
7. 750 v/cm 500 v/cm 0,541 ms 13 Ohm not used 60 NO
8. 750 v/cm 540 v/cm 0,523 ms 13 Ohm not used 60 5
9. 750 v/cm 510 v/cm 0,558 ms 13 Ohm not used 60 NO
10. 650 v/cm 430 v/cm 0,558 ms 13 Ohm not used 60 NO
11. 600 v/cm 400 v/cm 0,577 ms 13 Ohm not used 60 3
12. 550 v/cm 370 v/cm 0,535 ms 13 Ohm not used 60 4
13. 500 v/cm 320 v/cm 0,567 ms 13 Ohm not used 60 5
14. 500 v/cm 350 v/cm 0.531ms 13 Ohm not used 60 6
15. 500 v/cm 410 v/cm 3.690ms 129Ohm not used 60 NO
16. 500 v/cm 447 v/cm 13,10 ms 13Ohm 975 µF 96 NO
17. 500 v/cm 457 v/cm 13,21 ms 13Ohm 975 µF 100 NO
18. 500 v/cm 310 v/cm 0,535 ms 13 Ohm not used 60
500 v/cm 310 v/cm 0,530 ms 13 Ohm not used 2
19. 500 v/cm 320 v/cm 0,545 ms 13 Ohm not used 60
500 v/cm 320 v/cm 0,535 ms 13 Ohm notused 1

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20. 500 v/cm 340 v/cm 0,524 ms 13 Ohm not used 60
500 v/cm 330 v/cm 0,535 ms 13 Ohm not used NO
21. 500 v/cm 370 v/cm 0,459 ms 13 Ohm 25 µF 60
500 v/cm 366 v/cm 0,466 ms 13 Ohm 25 µF NO
22. 500 v/cm 452 v/cm 12,67 ms 13 Ohm 800 µF 60
500 v/cm 454 v/cm 12,69 ms 13 Ohm 800 µF NO
23. 400 v/cm 270 v/cm 0,562 ms 13 Ohm 50 µF 60 1
24. 350 v/cm 315 v/cm 5,330 ms 13 Ohm 400 µF 60
350 v/cm 320 v/cm 5,220 ms 13 Ohm 400 µF NO
25. 250 v/cm 206 v/cm 19,10 ms 13 Ohm 1600 µF 60 NO
26. 250 v/cm 190 v/cm 0,541 ms 13 Ohm not used 60 9
27. 250 v/cm 224 v/cm 13,00 ms 13Ohm 975 µF 60
250 v/cm 222 v/cm 12,96 ms 13Ohm 975 µF 2
28. 250 v/cm 218 v/cm 45,10ms 24 Ohm 1850 µF 60
250 v/cm 218 v/cm 46,30ms 24 Ohm 1900 µF
250 v/cm 218 v/cm 53,50ms 24 Ohm 2200 µF
250 v/cm 218 v/cm 48,90ms 24 Ohm 2100 µF
250 v/cm 218 v/cm 52,70ms 24 Ohm 2150 µF NO
*29. 250 v/cm 160 v/cm 0,584ms 13 Ohm 50 µF 60
250 v/cm 170 v/cm 0,573ms 13 Ohm 50 µF
250 v/cm 160 v/cm 0,583ms 13 Ohm 50 µF
250 v/cm 170 v/cm 0.572ms 13 Ohm 50 µF
250 v/cm 170 v/cm 0.575ms 13 Ohm 50 µF 4
30. 135 v/cm 104 v/cm 13,29 ms 13 Ohm 800 µF 60 2
31. 135 v/cm 102 v/cm 13,34ms 13 Ohm 975 µF 250 18
32. 125 v/cm 93 v/cm 43,30ms 13 Ohm 3175 µF 60
25 v/cm 15 v/cm 40,00ms 13 Ohm 3175 µF 2
33. 125 v/cm 105 v/cm 57,60 ms 48 Ohm 800 µF 60
25 v/cm 14 v/cm 41,50 ms 48 Ohm 800 µF 4
34. 125 v/cm 102 v/cm 12,50 ms 13 Ohm 800 µF 60
25 v/cm 13 v/cm 12,63 ms 13 Ohm 800 µF
25 v/cm 13 v/cm 12,70 ms 13 Ohm 800 µF NO
35. 125 v/cm 105 v/cm 42,60 ms 48 Ohm 800 µF 60
125 v/cm 103 v/cm 43,50 ms 48 Ohm 800 µF NO
36 50 v/cm 35 v/cm 47,10 ms 48 Ohm 800 µF 60 5
37. 50 v/cm 34 v/cm 12,37 ms 13 Ohm 800 µF 60
50 v/cm 35 v/cm 12,32 ms 13 Ohm 800 µF 10
38. 50 v/cm 36 v/cm 23,90 ms 24 Ohm 800 µF 60
50 v/cm 37 v/cm 23,11 ms 24 Ohm 800 µF 3
39. 50 v/cm 36 v/cm 46,90 ms 48 Ohm 800 µF 60
50 v/cm 35 v/cm 44,50 ms 48 Ohm 800 µF 2

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40. 50 v/cm 36 v/cm 39,80ms 48 Ohm 800 µF 60
50 v/cm 37v/cm 38,20ms 48 Ohm 800 µF
50 v/cm 36 v/cm 37,40ms 48 Ohm 800 µF
50 v/cm 37 v/cm 36,50ms 48 Ohm 800 µF
50 v/cm 35 v/cm 36,70ms 48 Ohm 800 µF NO
41. 50 v/cm 36 v/cm 12,26ms 13 Ohm 800 µF 60
50 v/cm 36 v/cm 12,25ms 13 Ohm 800 µF
50 v/cm 36 v/cm 12,24ms 13 Ohm 800 µF
50 v/cm 35 v/cm 12,60ms 13 Ohm 800 µF
50 v/cm 34 v/cm 12,57ms 13 Ohm 800 µF 6
42. 35 v/cm 21 v/cm 49,90 ms 48 Ohm 800 µF 60
35 v/cm 22 v/cm 45,50 ms 48 Ohm 800 µF
35 v/cm 21 v/cm 45,60 ms 48 Ohm 800 µF 2
*43. 25 v/cm 15 v/cm 43,30 ms 48 Ohm 800 µF 60
25 v/cm 15 v/cm 43,30 ms 48 Ohm 800 µF
25 v/cm 15 v/cm 42,60 ms 48 Ohm 800 µF
25 v/cm 14 v/cm 41,80 ms 48 Ohm 800 µF
25 v/cm 14 v/cm 41,30 ms 48 Ohm 800 µF 2
*44. 25 v/cm 14 v/cm 40,00ms 48 Ohm 800 µF 60
25 v/cm 14 v/cm 40,20ms 48 Ohm 800 µF
25 v/cm 15 v/cm 38,90ms 48 Ohm 800 µF
25 v/cm 15 v/cm 37,80ms 48 Ohm 800 µF
25 v/cm 15 v/cm 39,00ms 48 Ohm 800 µF NO
45. 25 v/cm 14 v/cm 51,50ms 24 Ohm 2125µF 60
25 v/cm 15 v/cm 50,20ms 24 Ohm 2125 µF
25 v/cm 16 v/cm 49,50ms 24 Ohm 2125 µF
25 v/cm 16 v/cm 50,10ms 24 Ohm 2125 µF
25 v/cm 16 v/cm 50,00ms 24 Ohm 2125 µF NO
*46. 25 v/cm 18 v/cm 63,90ms 48 Ohm 50 µF 60
25 v/cm 18 v/cm 35,60ms 24 Ohm 50 µF
25 v/cm 18 v/cm 35,60ms 24 Ohm 50 µF
25 v/cm 18 v/cm 35,90ms 24 Ohm 50 µF
25 v/cm 18 v/cm 35,20ms 24 Ohm 50 µF 6
* Here we used 80 µl electroporation buffer.

Table [2] Electroporation experiments using non-dechorionated embryos with Rad-Bio

cuvette 0,2cm with 40 µl electroporation buffer and DNA at concentration of 10 µg/ml.
Exp. Set V. Peak V. T.C. R. C. E.E. L.H.
1. 500 v/cm 320 v/cm 0,578ms 13 Ohm Not used 42 1
*2. 500 v/cm 340 v/cm 12,71ms 13 Ohm 800 µF 120
500 v/cm 340 v/cm 12,73ms 13 Ohm 800 µF 4
*3. 350 v/cm 320 v/cm 13,42ms 13 Ohm 975 µF 100 NO

20
*4. 250 v/cm 219 v/cm 13,32ms 13 Ohm 975 µF 98 3
5. 135 v/cm 104 v/cm 13,333ms 13 Ohm 800 µF 21 3
*6. 125 v/cm 90 v/cm 0,736ms 13 Ohm 50 µF 250 18
7. 125 v/cm 104 v/cm 12,78ms 13 Ohm 800 µF 80 6
8. 125 v/cm 88 v/cm 0,748ms 13 Ohm 50 µF 50
125 v/cm 97 v/cm 1,373ms 13 Ohm 100 µF 4
9. 100 v/cm 77 v/cm 1,390ms 13 Ohm 100 µF 60
100 v/cm 78 v/cm 1,389ms 13 Ohm 100 µF 8
10. 75 v/cm 68 v/cm 1,250ms 13 Ohm 100 µF 70
75 v/cm 68 v/cm 1,248ms 13 Ohm 100 µF 9
11. 75 v/cm 68 v/cm 12,54ms 13 Ohm 800 µF 60 7
12. 50 v/cm 48 v/cm 1,254ms 13 Ohm 100 µF 67
50 v/cm 47 v/cm 1,257ms 13 Ohm 100 µF 7
* Here we used 80 µl electroporation buffer.

Table [3] Electroporation experiments using non-dechorionated embryos with Petri dish
cuvette 0.1cm with 40µl electroporation buffer and DNA at concentration of 10µg/ml.
Exp. Set V. Peak V. T.C. R. C. E.E. L.L
1. 750 v/cm 540v/cm 0,531 ms 13 Ohm notused 34 NO
2. 500 v/cm 330 v/cm 0,571 ms 13 Ohm not used 90 5
3. 500 v/cm 330 v/cm 0,557 ms 13 Ohm not used 70
500 v/cm 320 v/cm 0,587 ms 13 Ohm not used 3
4. 500 v/cm 320 v/cm 0,587 ms 13 Ohm not used 44
500 v/cm 320 v/cm 0,586 ms 13 Ohm not used 2
5. 500 v/cm 330 v/cm 0,555 ms 13 Ohm not used 169
500 v/cm 320 v/cm 0,556 ms 13 Ohm not used 11
6. 350 v/cm 320 v/cm 13,02 ms 13 Ohm 975 µF 102 NO
7. 150 v/cm 118 v/cm 1,352 ms 13 Ohm 100 µF 95 15
8. 135 v/cm 86 v/cm 0,511 ms 13 Ohm 25 µF 100
25 v/cm 21 v/cm 0,448 ms 13 Ohm 25 µF 9
9. 135 v/cm 120 v/cm 10,10 ms 246Ohm 50 µF 35 NO
10. 125 v/cm 97 v/cm 1,365 ms 13 Ohm 100 µF 103 8
11. 125 v/cm 94 v/cm 19,10 ms 13 Ohm 1400 µF 70
25 v/cm 17 v/cm 15,30 ms 13 Ohm 1400 µF 3
12. 100 v/cm 82 v/cm 12,23 ms 13 Ohm 1000 µF 100
100 v/cm 83 v/cm 24,60 ms 13 Ohm 2000 µF 5

21
13. 100 v/cm 77 v/cm 1,390 ms 13 Ohm 100 µF 75 9
14. 100 v/cm 72 v/cm 13,66 ms 13 Ohm 800 µF 70 2
15. 90 v/cm 75 v/cm 3,560 ms 13 Ohm 250 µF 60 6
16. 75 v/cm 59 v/cm 1,397 ms 13 Ohm 100 µF 110 13
17. 75 v/cm 44 v/cm 0,758 ms 13 Ohm 50 µF 80 8
18. 65 v/cm 62 v/cm 6,260 ms 13 Ohm 500 µF 62 6
19. 50 v/cm 48 v/cm 1,260 ms 13 Ohm 100 µF 83 11
20. 50 v/cm 44 v/cm 0,677 ms 13 Ohm 100 µF 135 13
21. 50 v/cm 33 v/cm 13,43 ms 13 Ohm 975 µF 100 4

Table [4] Electroporation experiments using non-dechorionated embryos with different

cuvettes, 40 µl electroporation buffer and different DNA concentrations.
Exp. Set PeakV T.C. RΩ C.µF E.E. L.H. Adult Cuvette DNA
V. msec gap µg/ml
1. 500 455 12,44 13 875 110 6 4 BR2mm 10
2. 400 362 12,19 13 875 88 BR2mm 10
400 361 12,08 13 875 2 2
3. 400 369 3,79 72 50 85 1 NO Pd1mm 10
4. 350 323 3,54 72 50 115 3 1 Pd1mm 10
5. 300 285 3,54 72 50 100 7 2 Pd1mm 10
6. 250 223 23,6 24 975 50 NO NO Pd1mm 10
7. 250 229 3,52 72 50 50 7 2 Pd1mm 10
8. 200 183 3,51 72 50 85 11 4 Pd1mm 10
9. 200 188 10,12 129 100 88 8 2 Pd1mm 10
10. 150 135 3,54 13 50 50 9 3 Pd1mm 10
11. 145 129 3,54 72 50 80 5 NO Pd1mm 10
12. 50 50 8,88 720 50 55 10 4 Pd1mm 10
13. 50 34 12,46 13 975 80 BR2mm 10
50 34 12,26 13 975 18 11
*14. 50 34 11,79 13 975 100 BR2mm 10
50 32 12,32 13 975 23 18
*15. 500 468 20,7 129 400 130 NO NO BR2mm 20
16. 500 330 ,563 13 not 110 3 1 Pd1mm 100
used
17. 500 330 ,589 13 not 120 12 BR2mm 100
500 320 ,578 13 used 6
*18. 250 224 88,4 129 975 92 NO NO BR2mm 100
19. 200 175 4,79 48 50 105 8 6 Pd1mm 100

22
20. 125 94 13,12 13 975 100 15 6 Pd1mm 100
21. 100 83 12,18 13 975 83 11 7 Pd1mm 100
22. 50 47 45,10 129 500 100 10 5 Pd1mm 100
23. 50 46 131,6 720 500 92 5 2 Pd1mm 100
24. 50 51 9,23 129 100 93 11 Pd1mm 100
50 51 9.33 129 100 9
25. 250 216 13,01 13 975 100 NO NO Pd1mm 1000
26. 100 75 1,343 13 100 100 16 Pd1mm 1000
100 85 1,211 13 100 9
27. 35 20 12,2 13 975 90 18 14 BR1mm 1000
* Here we used 400 µg /ml electroporation buffer. BR, Bio-Rad cuvette. Pd, Petri dish
cuvette

Table [5] Electroporation experiments using non-dechorionated embryos with Petri dish
cuvette, 40 µl electroporation buffer and 20 µg/ml DNA concentrations
EXP. Set V. PeakV. T.C.msec. R.Ω C.µF E.E. L.H.
1. 575 357 ,566 13 not used 100 13
2. 500 350 ,575 13 not used 120 20
3. 500 320 ,570 13 not used 105 15
4. 400 270 ,573 13 not used 97 13
5. 250 180 ,577 13 50 35
250 180 ,585 13 50 8
6. 250 200 ,726 13 50 38
250 201 ,742 13 50 6
7. 170 170 3,67 129 50 54 4
8. 150 150 3,84 129 50 64 6
9. 150 140 2,00 48 50 76 10
10. 150 150 3,70 129 50 52 6
11. 140 140 3,99 129 50 80 16
12. 125 112 5,09 129 50 53 6
13. 125 88 ,725 13 50 48 10
14. 125 97 ,670 13 50 54 4
15. 125 88 ,743 13 50 100 20
16. 125 104 12,03 13 975 98 3
17. 100 93 11,65 720 50 89 4
18. 70 56 12,25 13 975 50 2
19. 65 49 12,16 13 25 87 15
20. 65 63 2,99 129 25 50 9
21. 65 66 14,05 720 50 120 14

23
22. 50 53 13,06 720 50 88 16
23. 35 38 2,36 13 175 54 13
24. 35 38 2,36 13 175 51
35 38 2,33 13 175 8
25. 35 39 5,67 129 50 100 9
26. 35 19 12,15 13 975 100 18
27. 30 27 7,59 13 575 60 9
28. 30 28 7,31 13 575 80 12
29. 25 25 7,18 13 575 80 13
30. 25 24 ,677 13 50 70 18
31. 25 23 7,38 13 575 30 4
*32. 25 28 1,218 13 1000 100 13
33. 20 20 ,680 13 50 45
20 20 ,675 13 50 11
34. 15 15 ,696 13 50 66
15 15 ,690 13 50 20
35. 10 13 ,687 13 50 80
10 13 ,702 13 50 25
●36. 10 14 ,693 13 50 60
10 14 ,689 13 50 17
37. 7 15 9,82 129 100 80 12
* Here we used 400 µg /ml DNA concentration with Bio-Rad cuvette 2mm.
● Here we used 1000 µg/ml DNA concentration.

Table [6] Micro-injection experiments using dechorionated GAL4-embryos micro-

injection with different DNA concentrations.
EXP. Embryos stock DNA concentration GFP signal
1. 4442 10 µg/ml _
2. 4442 10 µg/ml _
3. 4442 10 µg/ml _
4. 1973 10 µg/ml _
5. 4442 100 µg/ml _
6. 1973 100 µg/ml _
7. 4442 100 µg/ml _
8. 4442 1000 µg/ml +

24
Table [7] Electroporation experiments using dechorionated GAL4-embryos with Petri
dish cuvette, 40 µl electroporation buffer and 1000 µg/ml DNA concentrations
EXP. Set V. Peak V. T.C. R.Ω C.µF Stock GFP signal
msec.
1. 1250 940 , 146 13 not used 5460 _
2. 1250 940 , 149 13 not used 1973 _
3. 500 330 , 556 13 not used 4442 _
4. 350 250 , 566 13 50 1973 _
5. 350 250 , 578 13 50 5460
350 250 , 575 13 50 _
6. 350 250 , 569 13 50 4442 _
7. 350 240 , 617 13 50 4442
350 240 , 576 13 50 +
8. 350 329 4,45 129 50 4442 _
9. 175 131 , 726 13 50 4442
175 132 , 725 13 50 _
10. 100 88 40,5 129 475 1973 _
11. 100 90 40,2 129 475 1973 _
12. 35 20 12,20 13 975 4442
35 21 12,14 13 975 _
13. 35 33 7,26 13 575 1973 _
14. 35 33 7,16 13 575 1973 _
15. 35 34 7,18 13 575 5460 _
16. 35 33 7,13 13 575 5460 _
17. 35 33 7,24 13 575 4442 _
18. 35 33 7,34 13 575 4442 _
19. 25 25 7,20 13 575 5640 _
20. 25 30 14,75 720 50 1973
25 29 14,64 720 50 _

Table [8] Electroporation experiments using dechorionated ∆2-3-embryos with Petri dish
cuvette, 40 µl electroporation buffer and 1000 µg/ml DNA concentrations
Exp. SetV. PeakV. T.C.msc R.Ω C.µF E.E. L.H. Adult
1. 350 240 , 619 13 50 100
350 240 , 578 13 50 NO NO
2. 175 130 , 758 13 50 50
175 132 , 726 13 50 6 3
3. 150 109 , 741 13 50 60
150 110 , 736 13 50 7 3
4. 150 109 , 728 13 50 60

25
 150 110 , 719 13 50
150 110 , 726 13 50 3 NO
5. 125 89 , 739 13 50 70 9 5
6. 125 87 , 737 13 50 100 8 5
125 96 , 668 13 50
7. 100 88 39,0 129 475 100 NO NO
8. 100 89 37,1 129 475 60 NO NO
9. 100 88 44,2 129 475 110 NO NO
10. 35 20 12,47 13 975 90
35 20 12,57 13 975 6 2
11. 30 33 5,36 129 50 57
35 39 5,24 129 50 5 NO
12. 30 16 12,19 13 975 70
30 16 12,30 13 975 8 5
13. 25 12 12,69 13 975 44 6 4
14. 25 28 2,52 48 50 56
25 28 2,46 48 50 12 8
15. 20 20 6,19 13 500 60
20 20 6,34 13 500 11 5
16. 13 13 44,1 129 500 38 2 NO
17. 10 11 50,7 129 400 53
10 12 50,6 129 400 6 2
18. 5 14 44,1 720 175 60 2 1

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26
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