Embryo Microinjection for Transgenesis in Drosophila

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 effects and thereby can not be compared². These drawbacks were overcome by the phiC31-mediated site-specific transgenesis³. The bacteriophage phiC31 gene encodes an integrase that mediates sequence-specific recombination between the attB and attP sites in Drosophila³. Many attP docking sites have been characterized and are available in the Bloomington Drosophila Stock Center^{3,4,5,6,7,8,9}.

The phiC31 transgenesis system for Drosophila has been widely used by the fly community. Among the attP docking sites, attP18 on the X chromosome, attP40 on the second chromosome, and attP2 on the third chromosome are usually the preferred sites for transgene integration on each chromosome because transgenes at the three sites show high levels of inducible expression while having low levels of basal expression⁵. Recently, however, van der Graaf and colleagues found that the attP40 site, close to the Msp300 gene locus, and transgenes integrated at attP40 cause larval muscle nuclear clustering in Drosophila¹⁰. In another recent study, Duan and colleagues found that the homozygous attP40 chromosome disrupts the normal glomerular organization of the Or47b olfactory receptor neuron class in Drosophila¹¹. These findings suggest that rigorous controls should be included when designing experiments and interpreting data.

Drosophila is an ideal model organism for in vivo interrogation of gene function due to its short life cycle, low cost, and easy maintenance. Genome-wide genetic screening relies on the construction of genome-wide transgenic libraries in Drosophila^12,13,14,15. We previously generated 5551 UAS-cDNA/ORF constructs based on the binary GAL4/upstream activating sequence (GAL4/UAS) system, covering 83% of the Drosophila genes conserved in humans in the Drosophila Genomics Resource Center (DGRC) Gold Collection¹⁶. The UAS-cDNA/ORF plasmids can be used for the creation of a transgenic UAS-cDNA/ORF library in Drosophila. Efficient embryo microinjection technology can accelerate the creation of transgenic fly libraries.

For embryo microinjection, the needle tip is normally broken against a coverslip¹⁷. Thereby, needles frequently become clogged or cause leakage of large amounts of cytoplasm, and when these issues arise, new needles must be employed. Although beveling needles can enhance microinjection efficiency, the grinding solution enters the beveled tip during the grinding process. The grinding solution in the beveled tip does not quickly evaporate naturally; therefore, needles can not be utilized for microinjection directly after beveling. These factors reduce the efficiency of embryo microinjection procedures. Here, using the phiC31-mediated site-specific transgenesis in Drosophila as an example, we present a protocol for embryo microinjection for transgenesis in Drosophila. To prevent the grinding solution from entering the needle, we utilize an air pump to exert air pressure within the needle, thereby preventing the grinding solution from entering the beveled tip. Beveled needles are ready for sample loading and microinjection directly after beveling.

Protokół

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.
2. Determine the plasmid DNA concentration using a spectrophotometer and store it at -80 °C freezer.
   NOTE: Plasmid concentration is not critical for successful embryo microinjection, as plasmid concentrations ranging from 20 ng/µL to 1683 ng/µL work well for transgenesis in Drosophila (Supplementary Table 1).
3. Just before microinjection, thaw plasmids and centrifuge at 13000 x g for 5 min.
4. Transfer 10 µL of each plasmid from the upper layer of the liquid into a new 1.5 mL tube.

2. Pulling needles

1. Turn on a needle puller with selected settings (Heat: 500, Pull: 50, Vel: 60, Delay: 90, Pressure: 200, Ramp Test: 510).
2. Insert a borosilicate glass capillary (outer diameter 1.0 mm, inner diameter 0.75 mm, 10 cm length) into the needle puller and close the lid.
3. Press the Pull button. Each glass capillary makes two needles.

3. Grinding needles (Figure 1)

1. Use a needle grinder mounted with an appropriate abrasive plate (for 0.7-2.0 µm tip sizes) of a needle grinder.
2. Wet the cloth (reference wire) with grinding solution (100 mL of distilled water with 0.9 g of NaCl, 1 mL of wetting agent) and fix the cloth.
3. Turn on the needle grinder and keep the grinding plate rotating.
4. Connect a needle to a foot air pump with a pressure gauge via a silicone capillary tube, and then mount the needle onto a needle holder of the needle grinder. Adjust the angle of the needle relative to the grinding plate to 35° (Figure 1A, B).
5. Adjust the position of the needle tip just above the grinding plate surface by using a coarse control knob under the microscope (Figure 1C).
   NOTE: The pulled needles obtained in section 2 have a 6-8 mm taper and a less than 1 µm tip (Figure 1D), as is described in the user manual of the puller.
6. Keep the inner pressure of the needle at 40 psi using the foot air pump. Adjust the position of the needle tip by using a fine control knob under the microscope, and make the tip of the needle touch the grinding plate.
7. Grind for 3-5 s. When tiny air bubbles come out of the needle tip, unload the needle (Figure 1E).
   NOTE: In the process of grinding, using an air pump prevents the grinding solution from siphoning.
8. Put the ground needles into a storage case.
   NOTE: Use freshly ground needles as much as possible, though the ground needles can be kept in a storage case for at least a few weeks before microinjection.

4. Preparing pipettes for loading plasmids

1. Heat a 200 µL pipette with the outer flame of an alcohol burner.
2. Once it starts to melt, stretch the pipette.
3. Cut off the sealed end of the stretched pipette with scissors, and the stretched pipette is then ready to load DNA into a ground needle.
   NOTE: Alternatively, extremely long and fine tips for filling ground needles are commercially available.
4. Place these pipettes in an autoclaved plastic box.

5. Collecting embryos

1. Collect flies (vas-phiC31; + ; attP2) expressing phiC31 integrase and bearing the attP2 docking site 3 days after they are hatched from 50 vials and transfer them to a cage (φ 90 mm x 150 mm). Add a little fresh yeast paste to the center of each grape agar plate¹⁸.
2. Prepare 2-4 cages of flies. Keep the cages and plates at 25 °C and change the plates every day for 2-4 days.
   NOTE: This study combined the vas-phiC31 transgene (Stock # 36313, Bloomington Drosophila Stock Center) on the first chromosome and the attP2 transgene (Stock # 36313, Bloomington Drosophila Stock Center) on the third chromosome. The resulting stock (vas-phiC31; + ; attP2) is used in this protocol. Yeast paste needs to be prepared daily, and fresh yeast paste must be fed to the flies. The replaced cages need to be cleared and dried to get rid of the remaining embryos in the cages.
3. On the day of microinjection, change the cages and plates in the morning. Then, change the plates every 30 min and collect embryos from these plates. Change all the cages and plates at room temperature (RT) and afterward keep the cages and plates at 25 °C.
   NOTE: Some protocols prefer to incubate the cages and plates at 18 °C for egg laying. It depends on the number of embryos one needs for microinjection.

6. Dechorionating embryos

1. Prepare 30% bleach by mixing 60 mL of bleach (containing active chlorine content of 34-46 g/L) and 140 mL of double distilled water.
2. Remove the remaining flies on the grape juice agar plates with a brush or forceps, and rinse embryos into a cell strainer with double distilled water.
3. Dry the cell strainer containing the embryos with tissue paper.
4. Place it in a Petri dish containing 30% bleach, and swirl the cell strainer by hand or a horizontal shaker for 1.5 min at 60 rpm.
   NOTE: Rinsing is a critical step. If the rinsing time is not long enough, the shell of an embryo will not come off completely. If the rinsing time is too long, it will cause the embryo to become soft and break easily. The cell strainer should be completely submerged in the 30% bleach. Wear a pair of rubber gloves and a lab coat to avoid injuries caused by bleach.
5. Rinse embryos for 2 min with double distilled water stored in a laboratory plastic wash bottle to remove the residual bleach. Pick the floating embryos for the line-up.

7. Lining up embryos

1. Cut the solid grape juice agar into long strips using a blade.
   NOTE: Use purple grape juice to make it easier to line up white embryos.
2. Transfer the embryos onto a long strip using a paintbrush.
3. Line up around 60 embryos with a dissecting needle along the edge of a long strip with their posteriors facing the outside of the strip. Line up around 60 embryos within 5 min.
   NOTE: The micropyle is a cone-shaped protrusion at the anterior pole of the embryo, through which the sperm enters to fertilize the oocyte (Figure 2A). Therefore, based on the cone-shaped protrusion at the anterior pole of the embryo, it is easy to determine the posterior of the dechorionated embryo.
4. Place an 18 mm double-sided tape lengthwise along the edge of a coverslip (18 mm x 18 mm) and remove the paper backing. Gently press the coverslip onto the aligned embryos. Ensure the embryos are glued to the center of the tape.

8. Desiccating embryos

1. Place the coverslip with the lined-up embryos in a box containing desiccant. The drying time varies depending on temperature and humidity.
   NOTE: The drying time is critical for microinjection and needs to be experimentally determined. The drying time is usually 9-10 min in summer and 17-18 min in winter.
2. Cover the embryos with halocarbon oil.
   NOTE: Do not drop too much oil onto the embryos, as the extra oil may overflow the adhesive. The volume ratio of halocarbon oil 700 and halocarbon oil 27 is 1:3.

9. Microinjecting embryos

1. Load 2 µL of DNA into the needle by using the stretched pipette. Ensure that there are no bubbles in the needle. Place the needle into the needle holder of the micromanipulator.
2. Place the aligned embryos covered with halocarbon oil under an inverted microscope. Ensure that the posterior ends of the embryos face the needle.
3. Use the micromanipulator to bring the needle close to the embryos. Clear the needle. Ensure that a drop of liquid coming out of the needle is visible. Starting from 300 hPa injection pressure and 20 hPa equilibrium pressure, adjust the pressure of injection and the pressure of equilibrium to ensure the optimal size of the drop of liquid.
4. Use the micromanipulator to insert the needle into the posterior ends of the embryos, and use the foot control connected to the microinjector to inject the DNA (Figure 2). If a small halo is visible, DNA is successfully injected into the embryo.
   NOTE: Select the multinucleate embryos for injection and kill the older embryos using the "clean" button. If a large amount of cytoplasm leaks after injection, embryos are under-dried. If only a small amount of or no amount of cytoplasm leaks after injection, injections are considered successful. If more than one-third of the embryos leak cytoplasm after injection, extend the drying time by 1-2 min. If more than one-third of the embryos are over-dried, shorten the drying time by 1-2 min.
5. Remove the needle from the embryo. Move the needle to the posterior end of the next embryo. Repeat the above steps.
6. Put the injected embryos into an agar plate. Place the plate in a room at 18-20 °C. On the next day, transfer the plate to an incubator at a temperature of 25 °C and a humidity of 50%-60%, and incubate for 1 day.
   NOTE: After injection, keep embryos at a temperature of 18-20 °C to slow down their development, facilitate the integration of plasmid DNA into the Drosophila genome, and increase the survival rate of the injected embryos.

10. Collecting larvae

1. Use a dissecting needle to scrape and loosen the food in a vial. Supply the food with a few drops of distilled water.
   NOTE: Allow the distilled water to be absorbed into the food to prevent larvae from drowning.
2. Take out the plates from the incubator.
3. Collect the hatched larvae with a paintbrush under a stereo microscope and transfer them to the vial.
4. Place the vial into an incubator at a temperature of 25 °C and a humidity of 50%-60%.

11. Obtaining transgenic flies

1. Collect the hatched flies after the vial is incubated at 25 °C for 10 days.
2. Cross a single male fly to three virgin female flies of the TM2/TM6C Sb¹ Tb¹ balancers, and cross a single female fly to three male flies of the TM2/TM6C Sb¹ Tb¹ balancers.
3. Place the crosses into an incubator at a temperature of 25 °C and a humidity of 50%-60%.
4. Screen the progeny for flies with orange eyes. These are the transgenic flies (Figure 3A-C).
   NOTE: Flies bearing a copy of transgene at the attP2 docking site have orange eyes.
5. Cross a single orange-eyed male fly to three virgin female flies of the TM2/TM6C Sb¹ Tb¹ balancers to establish a stock.
   NOTE: More than one generation of crosses need to be set up in order to cross out the phiC31 transgene on the first chromosome.

Wyniki

The tip of an injection needle is beveled by a needle grinder (Figure 1). One can bevel 50-60 needles in one hour. DNA is microinjected into the posterior of an embryo at the pre-blastoderm or syncytial blastoderm stage, where nuclei share a common cytoplasm (Figure 2A). The posterior of an embryo can be readily located based on the micropyle at the anterior of the embryo (Figure 2A). Embryos at the cellular blastoderm and gastrulat...

Dyskusje

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...

Materiały

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