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
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.
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
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
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
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
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.
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 stocksThe 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. Theflies 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
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
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).
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 Figure-5 Bio-Rad Cuvettes
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.
For the control tests we used
GAL4-Drosophila melanogaster flies. These
Figure-7
A and B are two pieces of double sided
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).
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
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
Figure-8The thick piece of black textile wetted with fruit juice in the cap and the needle which we used.
Figure-9 Petri dish cuvette is designed to be watched under the microscope.
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.
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.
Some of the embryos died direct without showing any manifestations of growth. Others completed development into larva but they died at different instars. I have also noted that some larvae suffer from malformation in parts of the mouth and others 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
Figure-10
Positive result using DNA at concentration of 1000 µg/ml by micro-injection using dechorionated GAL4-embryos. A-arrow non-injectet embryo and B-arrow injected embryo.
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 method so it became apparent that the new
procedure of embryo preparation is better than the traditional procedure.
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
L R
Figure-11
Electroporated dechorionated GAL4-embryos in (R) right side and control embryos without electroporation in the (L) left side.
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.
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.
Tables
Tables abbreviations: V.; volt T.C.; time constant R.; resistance C.; capacitorE.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 250 v/cm 920 v/cm 170 v/cm 0,558 ms 0,599 ms 13 Ohm 13 Ohm not used not used 60 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 500 v/cm 310 v/cm 310 v/cm 0,535 ms 0,530 ms 13 Ohm 13 Ohm not used not used 60 2 19. 500 v/cm 320 v/cm 0,545 ms 13 Ohm not used 60
20. 500 v/cm
500 v/cm 340 v/cm 330 v/cm 0,524 ms 0,535 ms 13 Ohm 13 Ohm not used not used 60 NO 21. 500 v/cm 500 v/cm 370 v/cm 366 v/cm 0,459 ms 0,466 ms 13 Ohm 13 Ohm 25 µF 25 µF 60 NO 22. 500 v/cm 500 v/cm 452 v/cm 454 v/cm 12,67 ms 12,69 ms 13 Ohm 13 Ohm 800 µF 800 µF 60 NO 23. 400 v/cm 270 v/cm 0,562 ms 13 Ohm 50 µF 60 1 24. 350 v/cm 350 v/cm 315 v/cm 320 v/cm 5,330 ms 5,220 ms 13 Ohm 13 Ohm 400 µF 400 µF 60 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 250 v/cm 224 v/cm 222 v/cm 13,00 ms 12,96 ms 13Ohm 13Ohm 975 µF 975 µF 60 2 28. 250 v/cm 250 v/cm 250 v/cm 250 v/cm 250 v/cm 218 v/cm 218 v/cm 218 v/cm 218 v/cm 218 v/cm 45,10ms 46,30ms 53,50ms 48,90ms 52,70ms 24 Ohm 24 Ohm 24 Ohm 24 Ohm 24 Ohm 1850 µF 1900 µF 2200 µF 2100 µF 2150 µF 60 NO *29. 250 v/cm 250 v/cm 250 v/cm 250 v/cm 250 v/cm 160 v/cm 170 v/cm 160 v/cm 170 v/cm 170 v/cm 0,584ms 0,573ms 0,583ms 0.572ms 0.575ms 13 Ohm 13 Ohm 13 Ohm 13 Ohm 13 Ohm 50 µF 50 µF 50 µF 50 µF 50 µF 60 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 25 v/cm 93 v/cm 15 v/cm 43,30ms 40,00ms 13 Ohm 13 Ohm 3175 µF 3175 µF 60 2 33. 125 v/cm 25 v/cm 105 v/cm 14 v/cm 57,60 ms 41,50 ms 48 Ohm 48 Ohm 800 µF 800 µF 60 4 34. 125 v/cm 25 v/cm 25 v/cm 102 v/cm 13 v/cm 13 v/cm 12,50 ms 12,63 ms 12,70 ms 13 Ohm 13 Ohm 13 Ohm 800 µF 800 µF 800 µF 60 NO 35. 125 v/cm 125 v/cm 105 v/cm 103 v/cm 42,60 ms 43,50 ms 48 Ohm 48 Ohm 800 µF 800 µF 60 NO 36 50 v/cm 35 v/cm 47,10 ms 48 Ohm 800 µF 60 5 37. 50 v/cm 50 v/cm 34 v/cm 35 v/cm 12,37 ms 12,32 ms 13 Ohm 13 Ohm 800 µF 800 µF 60 10 38. 50 v/cm 50 v/cm 36 v/cm 37 v/cm 23,90 ms 23,11 ms 24 Ohm 24 Ohm 800 µF 800 µF 60 3 39. 50 v/cm 50 v/cm 36 v/cm 35 v/cm 46,90 ms 44,50 ms 48 Ohm 48 Ohm 800 µF 800 µF 60 2
40. 50 v/cm 50 v/cm 50 v/cm 50 v/cm 50 v/cm 36 v/cm 37v/cm 36 v/cm 37 v/cm 35 v/cm 39,80ms 38,20ms 37,40ms 36,50ms 36,70ms 48 Ohm 48 Ohm 48 Ohm 48 Ohm 48 Ohm 800 µF 800 µF 800 µF 800 µF 800 µF 60 NO 41. 50 v/cm 50 v/cm 50 v/cm 50 v/cm 50 v/cm 36 v/cm 36 v/cm 36 v/cm 35 v/cm 34 v/cm 12,26ms 12,25ms 12,24ms 12,60ms 12,57ms 13 Ohm 13 Ohm 13 Ohm 13 Ohm 13 Ohm 800 µF 800 µF 800 µF 800 µF 800 µF 60 6 42. 35 v/cm 35 v/cm 35 v/cm 21 v/cm 22 v/cm 21 v/cm 49,90 ms 45,50 ms 45,60 ms 48 Ohm 48 Ohm 48 Ohm 800 µF 800 µF 800 µF 60 2 25 v/cm 25 v/cm 25 v/cm 25 v/cm 15 v/cm 15 v/cm 15 v/cm 14 v/cm 43,30 ms 43,30 ms 42,60 ms 41,80 ms 48 Ohm 48 Ohm 48 Ohm 48 Ohm 800 µF 800 µF 800 µF 800 µF 60 *43. 25 v/cm 14 v/cm 41,30 ms 48 Ohm 800 µF 2 *44. 25 v/cm 25 v/cm 25 v/cm 25 v/cm 25 v/cm 14 v/cm 14 v/cm 15 v/cm 15 v/cm 15 v/cm 40,00ms 40,20ms 38,90ms 37,80ms 39,00ms 48 Ohm 48 Ohm 48 Ohm 48 Ohm 48 Ohm 800 µF 800 µF 800 µF 800 µF 800 µF 60 NO 45. 25 v/cm 25 v/cm 25 v/cm 25 v/cm 25 v/cm 14 v/cm 15 v/cm 16 v/cm 16 v/cm 16 v/cm 51,50ms 50,20ms 49,50ms 50,10ms 50,00ms 24 Ohm 24 Ohm 24 Ohm 24 Ohm 24 Ohm 2125µF 2125 µF 2125 µF 2125 µF 2125 µF 60 NO *46. 25 v/cm 25 v/cm 25 v/cm 25 v/cm 25 v/cm 18 v/cm 18 v/cm 18 v/cm 18 v/cm 18 v/cm 63,90ms 35,60ms 35,60ms 35,90ms 35,20ms 48 Ohm 24 Ohm 24 Ohm 24 Ohm 24 Ohm 50 µF 50 µF 50 µF 50 µF 50 µF 60 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
*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 125 v/cm 88 v/cm 97 v/cm 0,748ms 1,373ms 13 Ohm 13 Ohm 50 µF 100 µF 50 4 9. 100 v/cm 100 v/cm 77 v/cm 78 v/cm 1,390ms 1,389ms 13 Ohm 13 Ohm 100 µF 100 µF 60 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 50 v/cm 48 v/cm 47 v/cm 1,254ms 1,257ms 13 Ohm 13 Ohm 100 µF 100 µF 67 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
500 v/cm 330 v/cm 320 v/cm 0,557 ms 0,587 ms 13 Ohm 13 Ohm not used not used 70 3 4. 500 v/cm 500 v/cm 320 v/cm 320 v/cm 0,587 ms 0,586 ms 13 Ohm 13 Ohm not used not used 44 2 5. 500 v/cm 500 v/cm 330 v/cm 320 v/cm 0,555 ms 0,556 ms 13 Ohm 13 Ohm not used not used 169 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 25 v/cm 86 v/cm 21 v/cm 0,511 ms 0,448 ms 13 Ohm 13 Ohm 25 µF 25 µF 100 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 25 v/cm 94 v/cm 17 v/cm 19,10 ms 15,30 ms 13 Ohm 13 Ohm 1400 µF 1400 µF 70 3 12. 100 v/cm 100 v/cm 82 v/cm 83 v/cm 12,23 ms 24,60 ms 13 Ohm 13 Ohm 1000 µF 2000 µF 100 5
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
V. PeakV T.C. msec RΩ C.µF E.E. L.H. Adult Cuvette gap DNAµg/ml
1. 500 455 12,44 13 875 110 6 4 BR2mm 10 2. 400 400 362 361 12,19 12,08 13 13 875 875 88 2 2 BR2mm 10 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 50 34 34 12,46 12,26 13 13 975 975 80 18 11 BR2mm 10 *14. 50 50 34 32 11,79 12,32 13 13 975 975 100 23 18 BR2mm 10 *15. 500 468 20,7 129 400 130 NO NO BR2mm 20 16. 500 330 ,563 13 not used 110 3 1 Pd1mm 100 17. 500 500 330 320 ,589 ,578 13 13 not used 120 12 6 BR2mm 100 *18. 250 224 88,4 129 975 92 NO NO BR2mm 100
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 50 51 51 9,23 9.33 129 129 100 100 93 11 9 Pd1mm 100 25. 250 216 13,01 13 975 100 NO NO Pd1mm 1000 26. 100 100 75 85 1,343 1,211 13 13 100 100 100 16 9 Pd1mm 1000 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. 250 250 180 180 ,577 ,585 13 13 50 50 35 8 6. 250 250 200 201 ,726 ,742 13 13 50 50 38 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
22. 50 53 13,06 720 50 88 16 23. 35 38 2,36 13 175 54 13 24. 35 35 38 38 2,36 2,33 13 13 175 175 51 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 20 20 ,680 ,675 13 13 50 50 45 11 34. 15 15 15 15 ,696 ,690 13 13 50 50 66 20 35. 10 10 13 13 ,687 ,702 13 13 50 50 80 25 ●36. 10 10 14 14 ,693 ,689 13 13 50 50 60 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 +
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.
msec. R.Ω C.µF Stock GFP signal 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. 350 350 250 250 , 578 , 575 13 13 50 50 5460 _ 6. 350 250 , 569 13 50 4442 _ 7. 350 350 240 240 , 617 , 576 13 13 50 50 4442 + 8. 350 329 4,45 129 50 4442 _ 9. 175 175 131 132 , 726 , 725 13 13 50 50 4442 _ 10. 100 88 40,5 129 475 1973 _ 11. 100 90 40,2 129 475 1973 _ 12. 35 35 20 21 12,20 12,14 13 13 975 975 4442 _ 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 25 30 29 14,75 14,64 720 720 50 50 1973 _
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. 350 350 240 240 , 619 , 578 13 13 50 50 100 NO NO 2. 175 175 130 132 , 758 , 726 13 13 50 50 50 6 3 3. 150 150 109 110 , 741 , 736 13 13 50 50 60 7 3 4. 150 109 , 728 13 50 60
150 150 110 110 , 719 , 726 13 13 50 50 3 NO 5. 125 89 , 739 13 50 70 9 5 6. 125 125 87 96 , 737 , 668 13 13 50 50 100 8 5 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 35 20 20 12,47 12,57 13 13 975 975 90 6 2 11. 30 35 33 39 5,36 5,24 129 129 50 50 57 5 NO 12. 30 30 16 16 12,19 12,30 13 13 975 975 70 8 5 13. 25 12 12,69 13 975 44 6 4 14. 25 25 28 28 2,52 2,46 48 48 50 50 56 12 8 15. 20 20 20 20 6,19 6,34 13 13 500 500 60 11 5 16. 13 13 44,1 129 500 38 2 NO 17. 10 10 11 12 50,7 50,6 129 129 400 400 53 6 2 18. 5 14 44,1 720 175 60 2 1 ▓
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