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Nov 2020
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En masse DNA Electroporation for in vivo Transcriptional Assay in Ascidian Embryos
海鞘胚胎中DNA电穿孔的体内转录分析    

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Abstract

Ascidian embryos are powerful models for functional genomics, in particular, due to the ease of generating a large number of transgenic embryos by electroporation. In addition, the small size of their genome makes them an attractive model for studying cis-regulatory elements that control gene expression during embryonic development. Here, I describe the adaptation of the seminal method developed 25 years ago in Ciona robusta for en masse DNA electroporation for in vivo transcription to additional species belonging to three genera. It is likely that similar optimizations would make electroporation successful in other ascidian species, where in vitro fertilization can be performed on a large number of eggs.

Keywords: Ascidians (海鞘), Tunicates (被囊动物), Electroporation (电穿孔), Transgenesis (转基因技术), Cis-regulatory DNA (DNA顺式), Phallusia (海鞘属), Ascidia (海鞘), Molgula (莫古拉)

Background

Ascidians are marine invertebrates that are the closest vertebrate relatives. Their fast, stereotyped, and external embryonic development gives rise to tadpole-like larvae closely resembling other chordates (vertebrates and amphioxus). Their compact genome, together with the availability of simple and efficient methods to manipulate gene expression and function, have made ascidians interesting model organisms for functional genomics (Lemaire, 2011; Satoh, 2014). In particular, the introduction of plasmid DNA into a fertilized egg by electric shock (electroporation) allows the generation of hundreds to thousands of transient transgenic embryos, which promoted Ciona robusta as the reference ascidian species almost 25 years ago (Corbo et al., 1997). DNA electroporation is a widely used method for in vivo transcriptional assays (for characterization of the activity of candidate cis-regulatory elements using reporter genes), over-expression/knockdown, and live imaging.


Ascidians are a diverse group of animals containing around 3,000 species that have been proven as interesting systems to study the evolution of developmental mechanisms since their genomes have been extensively rearranged, but their embryonic development is strongly conserved (Dardaillon et al., 2020). In particular, DNA electroporation has been applied to various species (Roure et al., 2014; Stolfi et al., 2014; Colgan et al., 2019; Coulcher et al., 2020). Here, I provide a detailed protocol to perform electroporation in four species: Phallusia mammillata, Phallusia fumigata, Ascidia mentula, and Molgula appendiculata (Figure 1), which cover almost 400 million years of evolution.

Materials and Reagents

  1. Disposable scalpels

  2. 300- and 120-µm homemade sieves

    Note: Cut a 50-ml plastic tube at ~4-5 cm from the opening. Cover a heating plate with aluminum foil. Fuse a piece of nylon mesh (Sefar Nitex, catalog numbers: 03-300/51 [300-µm opening], 03-120/49 [120-µm opening]) to the cut tube using the heated plate.

  3. 15-ml glass centrifuge tubes (Dutscher, catalog number: 092305)

  4. 15-ml plastic conical tubes (e.g., Sarstedt, catalog number: 62.554.502)

  5. 60-mm plastic Petri dishes (e.g., Sarstedt, catalog number: 82.1194.500)

  6. 92-mm plastic Petri dishes (e.g., Sarstedt, catalog number: 82.1473.001)

  7. 60-mm and 92-mm agarose-coated Petri dishes. Alternatively, gelatin-coated dishes (GF) can be used as in Sardet et al. (2011)

    Note: Melt the agarose in sea water (1 g per 100 ml sea water). Fill a 92-mm dish with the hot solution. Make a thin agarose layer by pouring the agarose solution into the next dish; repeat until you have enough dishes (you will need two dishes per electroporation, plus a dozen for dechorionation, fertilization, and washing).

  8. 6-well plates

    Note: Eggs tend to stick to new plastic material. To avoid this, reuse the same plates; simply wash with tap water and air dry.

  9. Gloves (Phallusia blood is a powerful stain!)

  10. Glass Pasteur pipets

    Note: Soak them in tap water to avoid sticking of the embryos. Smooth the opening using a lighter. Prepare some with a larger opening (2-3 mm) using a diamond for egg collection in Phallusia.

  11. 100 ml glass beaker

  12. Horizontal rotating shaker (e.g., VWR, catalog number: 444-2900)

  13. 1.5 ml microtubes

  14. Low-binding 1.5 ml (e.g., VWR, catalog number: 525-0230) and 2 ml (e.g., VWR, catalog number: 525-0232) microtubes

  15. 4 mm electroporation cuvettes (e.g., Dutscher, catalog number: 38191)

  16. Depression slides: 4 mm (Dutscher, catalog number: 065230) and 1.5 mm (Dutscher, catalog number: 65227) of thickness

  17. Tungsten needle (Roboz Surgical Instrument Co., catalog number: RS-6065)

  18. Adult ascidians are collected from the ocean by diving, using a trawl or a dredge and are provided by the French node of the European research infrastructure EMBRC (Station biologique de Roscoff: Phallusia mammillata and Ascidia mentula; Station marine de Banyuls-sur-mer: Phallusia mammillata, Phallusia fumigata, Ascidia mentula, and Molgula appendiculata)

  19. Supercoiled circular plasmid DNA (20-100 µg) of the reporter construct

  20. NaOH, 2.5 M

  21. NaOH, 1 M

  22. 1 M Tris, pH 9.5

  23. 0.96 M D-mannitol solution (VWR, catalog number: 25311.297)

  24. TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)

  25. 25% glutaraldehyde solution (Sigma-Aldrich, catalog number: G6257)

  26. Sodium thioglycolate (Sigma-Aldrich, catalog number: T0632)

  27. Pronase (Sigma-Aldrich, catalog number: P5147)

  28. 0.2 µm filtered natural sea water or artificial sea water (BASWH: see Recipes)

  29. Phallusia and Ascidia dechorionation solution (see Recipes)

  30. Molgula dechorionation solution (see Recipes)

  31. PBTw (see Recipes)

  32. 10× PBS (see Recipes)

  33. X-gal staining solution (see Recipes)

  34. X-gal stock solution (see Recipes)

Equipment

  1. Scissors and tweezers

  2. Temperature-controlled room set at 18°C

  3. Dissecting scope for live embryo manipulation (e.g., Discovery V8 from Zeiss)

  4. Square wave electroporator (Harvard Apparatus, BTX ECM830, catalog number: 45-0052)

  5. Incubators

  6. Dissecting scope equipped with a digital camera for staining analysis (e.g., Discovery V20+AxioCam Erc 5s from Zeiss)

Procedure

The in vivo transcriptional assay proceeds according to these successive steps: 1) gamete collection, 2) egg dechorionation, 3) fertilization, 4) DNA electroporation, 5) embryo fixation and staining, and 6) data collection and analysis. The first 4 steps differ between species. Here, I present two versions: one for P. mammillata, P. fumigate, and A. mentula (with minor changes among species); and one for M. appendiculata.



Figure 1. Adult ascidian species. (A) Phallusia mammillata (5-20 cm long). (B) Phallusia fumigata (5-20 cm long). (C) Ascidia mentula (4-10 cm long). (D) Molgula appendiculata (3-6 cm long).


  1. Gamete collection

    Ascidians are hermaphrodites. The aim is to collect both eggs and gametes, separately, from each individual.

    1. Phallusia and Ascidia

      P. mammillata, P. fumigate, and A. mentula belong to the same family, the Phlebobranchia. Gametes are collected from the gonoducts by dissection. To avoid unwanted self-fertilization, eggs are collected first. All species have transparent eggs.

      1. Open the animals with a scalpel by cutting between the two siphons (Figure 2A). Phallusia tunic is thick and hard.

      2. Incise the tunic toward the base of the animal on both sides. Do not cut too deep to avoid damaging the animal.

      3. Open the tunic by pulling apart with your fingers.

      4. Transfer the animal to a dish on its left side (you should see the heart beating at the base of the animal, opposite to the siphons). If the animal is on the right side, you should see a large, yellowish (Phallusia, Figure 2C) or reddish (Ascidia, Figure 2B) oviduct full of eggs.

      5. Make a small cut in the oviduct and collect the eggs with a Pasteur pipet (Figure 2D).

        Note: The eggs in the oviduct are very compact and embedded in a gelatinous substance. Use a wide-bore Pasteur pipet for Phallusia; otherwise, their aspiration through the small opening of a pipet may cause extreme deformation and egg death. Dead eggs are easily spotted since they turn opaque.



        Figure 2. Gamete collection in Phallusia and Ascidia. (A) Schematic diagram illustrating the successive incisions (numbered red dotted lines) to open the tunic (left: lateral view with the two siphons pointing to the left; right: side view with the two siphons pointing toward the experimenter). (B) Dissected A. mentula individual where the gonoducts are exposed. (C) Dissected P. mammillata individual where the oviduct is clearly visible. (D) Egg collection in P. mammillata using a wide-bore Pasteur pipet. (E) P. mammillata egg. (F) A. mentula egg. Scale bar: 100 µm in D and E.


      6. Transfer the eggs to a 6-well plate filled with sea water. Phallusia individuals normally have a high number of eggs (0.5-2 ml eggs). Ascidia usually have fewer eggs, but the amount can reach 0.5-1 ml.

      7. Check the egg quality (transparent and uniform egg cytoplasm, Figures 2E and 2F) and transfer them to a glass tube for several washes with sea water. Eggs can be kept overnight at 14°C, spread on a Petri dish, with no major decrease in quality.

      8. You should see the spermiduct (sharp white duct) underneath the oviduct once you have removed the eggs. Make a small cut and collect the sperm into a 1.5-ml tube using a Pasteur pipet for Phallusia or a micropipet with a yellow tip. Avoid collecting sea water, debris, and blood. Dry sperm remains active for several days at 4°C.


    2. Molgula

      Contrary to Phallusia and Ascidia, very few eggs are present in the tiny oviduct (Figure 3D). Gametes are therefore collected by gonadal dissection. The oocytes collected from the gonad represent all stages of oogenesis. The procedure aims at collecting mainly fully grown oocytes that are not yet mature, as evidenced by the presence of a large germinal vesicle (GV). Fortunately, these oocytes mature spontaneously in sea water, and germinal vesicle breakdown (GVBD) occurs within 30-60 min.

      1. Open the animals with scissors, starting from one siphon around the animal all the way to the other siphon (Figure 3A) (M. appendiculata are covered with debris, shells, and stones; use large scissors and make your way around these obstacles).

      2. Pull open the tunic and drag the animal still attached by the siphons (Figure 3B).

      3. Cut the body wall from siphon to siphon with fine scissors and pull open the animal (Figure 3C).

      4. Under the dissecting scope, locate the gonads (one on each side of the animal, Figure 3C) under the pharyngeal basket with tweezers. You should see the short gonoducts pointing toward the siphons and the large ovary covered on one side by the testis (Figure 3D).

      5. Cut out each gonad with scissors and place them in the same well of a 6-well plate filled with sea water (Figure 3E).



        Figure 3. Gamete collection in Molgula appendiculata. (A) Schematic diagram illustrating the incision (red dotted line) to perform to open the tunic (left: lateral view with the two siphons pointing to the top; right: side view). (B) An individual (right side) after tunic (left side) removal. (C) Cut open the animal with a gonad visible at the center on each side. (D) Each gonad is composed of an ovary surrounded by a testis. The inlet shows a close-up view of the tiny spermiduct (top) and the oviduct (bottom), both openings pointing to the right. (E) A 6-well plate where pairs of gonads from 4 individuals have been collected. (F) A fertilizable egg is obtained after spontaneous maturation of a fully grown oocyte (scale bar: 50 µm).


      6. Under the dissecting scope, release the oocytes from the ovary using tweezers, and transfer the testis to a Petri dish filled with sea water (60-mm diameter). You should see a full range of oocytes (from tiny transparent oocytes to large, opaque, fully grown oocytes with GV).

      7. Transfer all the oocytes from one individual (2 ovaries) through a 300 µm sieve placed in a 100 ml glass beaker (gonad debris will not go through).

      8. Transfer the contents of the beaker to a 120 µm sieve and wash extensively with sea water to remove sperm and small oocytes.

      9. Transfer the oocytes to a 60 mm agarose-coated dish.

      10. Wait 30-60 min until GVBD occurs and produces fertilizable oocytes.

      11. Release the sperm from the testis of all individuals that have been collected in the same Petri dish by dissociating each testis with tweezers (discard the testes afterwards). Collect the concentrated sperm solution into a 15-ml plastic tube and keep at 4°C until use.


  2. Egg dechorionation

    The chorion that protects the egg is made of an inner vitelline membrane consisting of extracellular material and an outer cellular layer of follicle cells. To perform electroporation, it is necessary to remove it; this is achieved using a mixture of sodium thioglycolate and pronase. The concentration of each compound and the duration of the dechorionation varies between species.

    Note: Dechorionation is usually performed before fertilization, and the naked eggs can still be fertilized. For Molgula, since the procedure is rather quick, this can be performed after fertilization.


    1. Collect the eggs in a glass tube and allow them to settle to the bottom.

    2. Discard the excess sea water.

    3. Activate the dechorionation solution by raising the pH (add 3 drops (around 100 µl) (Phallusia/Ascidia) or 6 drops (around 200 µl) (Molgula) 2.5 M NaOH to 7 ml dechorionation solution using a Pasteur pipet, and mix).

    4. Proceed to dechorionation

      1. In an agarose-coated dish for Phallusia/Ascidia, by adding the eggs to the dechorionation solution (~7 ml for a 60-mm dish and ~14 ml for a 92-mm dish). Mix well and place on a horizontal shaker at ~70 rpm.

      2. In a glass tube for Molgula, by adding 3-4 ml dechorionation solution. Mix well by pipetting up and down.

    5. Regularly check the dechorionation status under the scope. Dechorionation takes 30-45 min (Phallusia/Ascidia) and 7-15 min (Molgula) depending on the egg density.

    6. Stop dechorionation when most eggs are clearly devoid of the chorion. Phallusia/Ascidia: gather the eggs at the center by swirling the dish and transfer them to a 15 ml glass tube. Add sea water to the top and gently mix by pipetting up and down. Molgula: discard as much dechorionation solution as possible. Add sea water to the top and gently mix by pipetting up and down.

      Note: Dechorionation is more difficult to follow in Molgula: while follicle cells are quickly lost, the vitelline membrane is more difficult to see (look closely, change the scope mirror orientation; the presence of test cells on the egg’s surface is a good indication that the eggs are not fully dechorionated).

    7. Wash extensively 2-4 times by allowing the eggs to settle down, replacing the sea water, and gently mixing with a Pasteur pipet. Debris (chorion and dead eggs) will float, while naked eggs rapidly sink to the bottom of the tube. Naked eggs are fragile and explode easily; be gentle and avoid air bubbles.

    8. You can proceed to the next step using the same tube or collect the eggs in an agarose-coated dish for later use. Dechorionated, unfertilized eggs can be stored overnight at 4-14°C with no major decrease in quality.


  3. Fertilization

    1. (Phallusia/Ascidia only) Add 5 µl dry sperm to a 1.5-ml microtube containing 1 ml sea water.

    2. (Optional) Fertilization is usually efficient with straight sperm solution; however, to achieve a complete fertilization rate and high synchrony, it is better to activate the sperm solution by raising the pH (in the ocean, sperm are activated by the slightly basic pH of the sea water). Add either 50 µl 1 M Tris pH 9.5 (Phallusia), 4 µl 1 M NaOH (Ascidia), or 8 µl 1 M NaOH (Molgula) to the sperm solution.

      Note: Phallusia sperm can also be activated by incubating the 1 ml mixture for 15 min with 20 µl chorionated eggs as in Sardet et al. (2011).

    3. Check the sperm motility by diluting an aliquot 50-100× in sea water and looking under the dissecting scope at high magnification. Sperm should be swimming frantically.

    4. Add ~200 µl (60-mm dish) or 400-500 µl (92-mm dish or 15-ml glass tube) sperm solution.

    5. Mix well, making the eggs float in the medium. You should see the sperm swimming intensively.

    6. Check for egg deformation that occurs following fertilization and that should be visible within the first few minutes.

    7. At 10-15 min post-fertilization, proceed to the washes to remove the sperm, since this protocol uses enormous quantities of sperm. (Fertilization in a Petri dish) Gather the eggs at the center by swirling the dish and transfer them to a 15-ml glass tube using a Pasteur pipet. Allow the eggs to settle to the bottom of the tube, discard most of the sea water using a Pasteur pipet, add clean sea water up to the top by gently pouring down the side of the tilted tube; resuspend the eggs thoroughly by flushing sea water with the pipet. Repeat this wash 1-3 ×.

      Note: Embryos without their protective chorion are fragile and explode as soon that they come into contact with the water/air interface. Be cautious; gently manipulate them, avoiding air bubbles.


  4. DNA electroporation

    1. Prepare the DNA (20-100 µg) in 50 µl TE.

    2. Add 200 µl 0.96 M D-mannitol and mix well.

    3. Collect the fertilized eggs at the bottom of a glass tube.

    4. Mark with a pen, the 100 µl level on the low-binding 1.5 ml tubes.

    5. Transfer the fertilized eggs into the 1.5-ml low-binding tubes. Try to add the same number of eggs to each tube (one per construct to be tested).

    6. Adjust the volume to the 100 µl mark with sea water.

    7. To each tube, add 250 µl DNA/mannitol solution.

      Note: Mannitol is used to reduce the salt concentration (and thus avoid electric arching during electroporation) while keeping the osmolarity at a sufficient level.

    8. Transfer the total mixture to an electroporation cuvette (use a different Pasteur pipet for each tube to avoid mixing DNA).

    9. Place the cuvette into the electroporator cuvette holder.

    10. Apply a single electrical pulse of 37 V (Phallusia/Ascidia) or 20 V (Molgula) for 32 ms. The pulse should be done toward the end of the first cell cycle (but before cleavage): at 50-60 min post-fertilization.

      Note: Electroporation can be performed any time after fertilization, and the timing does not affect the electroporation efficiency; however, the fertilized eggs are more robust to electroporation (but also to microinjection) during the last third of the first cell cycle.

    11. Proceed to the electroporation of the next construct.

    12. Once all electroporations are done, add some sea water to the cuvette and transfer the eggs to 92 mm agarose-coated Petri dishes (usually at least two dishes for one electroporation).

      Note: A total of 4-6 electroporations are routinely done per fertilization round. With experience, 10-12 electroporations can be performed.

    13. Check the dish under the dissecting scope. You should see intact embryos (since they are toward the end of the first cell cycle, you should see the myoplasm and/or deformations corresponding to the preparation of the first cleavage) and debris of exploded eggs.

      Note: For species with transparent eggs (Phallusia/Ascidia), dead eggs are easily spotted since they become opaque. In most cases, there is a small proportion of eggs that do not survive the electroporation; these correspond to fragile or unfertilized eggs. In cases where you find no surviving embryos, there are two main explanations: the fertilization failed (unfertilized eggs systematically explode after the electrical pulse) or the voltage was too high. Try to make two experimental controls: non-dechorionated and non-electroporated embryos, and dechorionated and non-electroporated embryos.

    14. Transfer the dishes to an incubator set at the desired temperature.

      Note: 14-19°C is the common range of temperature for all species. They can also develop well at lower temperatures with a significant decrease in speed. Phallusia/Ascidia also develop well up to 22°C.

    15. Spread the embryos by flushing sea water with a Pasteur pipet (they have a very high tendency to stick to each other if they are too close). Avoid moving the dishes before the 8-cell stage since blastomeres are very loosely attached to each other.


  5. Embryo fixation and staining

    Depending on the reporter gene, the course of action may differ. Here, I describe the use of LacZ as a reporter gene (detecting β-galactosidase activity using the chromogenic substrate X-gal).

    1. Allow the embryos to develop until the desired stage.

    2. Swirl the dish to gather the embryos at the center.

    3. Collect the embryos using a glass Pasteur pipet and transfer them to a 2-ml low-binding microtube.

    4. Allow the embryos to settle and discard the excess sea water.

    5. Fill the tube with fixative (0.2% glutaraldehyde in sea water) and rotate the tube in your fingers for 30 s.

    6. Fix for exactly 30 min at room temperature (over-fixing will abolish β-galactosidase enzymatic activity, too light a fixation may lead to embryo disintegration).

    7. Remove half of the fixative and add 1 ml PBTw. Mix by inverting 2-3 ×.

    8. Once the embryos have settled, replace the solution with 2 ml PBTw, mix by inversion, and wait 10 min. Repeat this wash 1 ×.

    9. Rinse for 5 min in X-gal staining solution.

    10. Replace with 250 µl staining solution containing 0.4 mg/ml X-gal (dilute the X-gal stock solution 100×).

    11. Incubate at 37°C (an incubator is preferable to a water bath since no evaporation takes place in the tube).

    12. Check the blue staining from time to time (it starts within a couple of hours depending on the strength of the tested regulatory region).

    13. Once the staining is adequate (this may take several days, but this is a staining procedure that never produces any background), wash 2 × 5 min in PBTw. Typical examples of staining with a version of β-galactosidase targeted to the nucleus may be found in Roure et al. (2014) and Coulcher et al. (2020).

    14. Post-fix for 1 h at room temperature or O/N at 4°C in PBTw containing 3.7% formaldehyde.

    15. Samples can be stored long-term.

Data analysis

  1. Data collection

    1. Discard the fixative and wash 2 × 10 min in PBTw.

    2. Transfer all the embryos to a thick depression slide.

    3. Transfer part of the embryos to a thin depression slide.

    4. Using a dissecting scope and a tungsten needle, score the number of stained embryos in the target tissue until you reach 100-200 scored embryos or until all the embryos in the tube have been scored (score only those embryos that have developed normally) (Roure et al., 2014; Coulcher et al., 2020).

      Notes:

      1. Embryos can be manipulated easily using a tungsten needle, a cat whisker, or an eyelash.

      2. Depending on the aim, the approach can be refined by scoring not only the number of stained embryos, but also the number of stained cells per embryo; however, this may be time consuming.

      3. Do not forget that this transgenesis method yields mosaic transgene expression (each cell has randomly inherited a variable amount of plasmid DNA) and that each embryo is an independent event; hence, the activity of a given region is determined by compiling the expression in all the embryos that are analyzed.

    5. Take images of representative embryos.


  2. Evaluation of cis-regulatory activity and comparisons

    1. The assay provides qualitative data (i.e., the spatial domain of activity of a given piece of genomic DNA), but the percentage of stained embryos also reflects the strength of the regulatory region.

    2. The score obtained may vary from experiment to experiment according to the ascidian batch, plasmid DNA preparation, and embryo concentration during electroporation; thus, we usually perform at least 3 biological replicates (different ascidian, different day of experimentation) and calculate an average score and standard deviation.

      Note: Long (4-10 kb) genomic regions are usually strongly active (staining is visible within 1 h of incubation at 37°C, with over 90% of the embryos stained) and do not display much variation between experiments.

    3. Often, the goal of this assay is to locate essential cis-regulatory elements; thus, one needs to compare the activity of various constructs. Obviously, the plasmid backbone should be the same. We also favor comparing the results of constructs that have been electroporated during the same experiment.

Recipes

  1. Banyuls-sur-mer Artificial Sea Water HEPES (BASWH)

    BASWH is a ‘Mediterranean type’ sea water with the following features: salinity of 37.9 g/L, osmolality of 1219 mOsmole, Na+/K+ ratio of 47, and a pH of ~8.0.

    For 1 L, mix:

    30.34 g NaCl

    0.83 g KCl

    1.47 g CaCl2·2H2O

    4.98 g MgCl2·6H2O

    6.29 g MgSO4·7H2O in MilliQ water

    Adjust to 1 L

    At this step, the solution can be stored at room temperature for months.

    Before use, add 0.18 g NaHCO3 and 5 ml HEPES 1 M pH 8.0, and filtrate (0.2 µm). Can be stored for 1-2 months at 4°C.

  2. Phallusia and Ascidia dechorionation solution

    1% sodium thioglycolate

    0.05% pronase in sea water

    Mix thioglycolate in sea water by shaking, add pronase on top – no mixing – and keep at 4°C for a few hours before use.

    Can be used for 1-2 weeks when kept at 4°C.

    Bring aliquot to room temperature before use.

  3. Molgula dechorionation solution

    0.5% sodium thioglycolate

    0.1% pronase in sea water

    Mix thioglycolate in sea water by shaking, add pronase on top – no mixing – and keep at 4°C for a few hours before use.

    Can be used for 1-2 weeks when kept at 4°C.

    Bring aliquot to room temperature before use.

  4. PBTw

    PBS 1× containing 0.1% Tween-20

  5. PBS 10×

    For 1 L, add:

    80.0 g NaCl

    26.8 g Na2HPO4·7H2O

    2.0 g KCl

    2.4 g KH2PO4

    Adjust pH to 7.4 with NaOH

    Bring to 1 L, and autoclave

  6. X-gal staining solution

    1 mM MgCl2

    3 mM K4Fe(CN)6

    3 mM K3Fe(CN)6 in PBTw

  7. X-gal stock solution

    40 mg/ml X-gal in dimethylformamide

Acknowledgments

This protocol was used in the study ‘Conservation of peripheral nervous system formation mechanisms in divergent ascidian embryos’ by Coulcher et al. published in eLife in 2020 (doi: 10.7554/eLife.59157). This work was made possible by provision of adult ascidians thanks to the knowledge and generosity of G. Diaz (professional fisherman in Port-Vendres, France) and staff (divers and boat crew) at the marine stations of Banyuls-sur-mer and Roscoff (French node of the European research infrastructure EMBRC). I wish to thank members of my group. This work was supported by CNRS and Sorbonne Université, and by specific grants from the ANR (ANR-11-JSV2-007 and ANR-17-CE13-0027), the CNRS (DBM2020 from INSB), and the European project Assemble Plus (H2020-INFRAIA-1-2016–2017; Grant No. 730984).

Competing interests

There are no conflicts of interest or competing interests.

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  8. Satoh, N. (2014). Developmental genomics of ascidians. Hoboken, New Jersey, John Wiley & Sons, Inc.
  9. Stolfi, A., Lowe, E. K., Racioppi, C., Ristoratore, F., Brown, C. T., Swalla, B. J. and Christiaen, L. (2014). Divergent mechanisms regulate conserved cardiopharyngeal development and gene expression in distantly related ascidians. Elife 3: e03728.

简介

[摘要]海鞘胚胎是用于功能基因组学强大的模型,特别是,由于容易生成的一个通过电穿孔大量转基因胚胎的。此外,它们的基因组化妆的小尺寸š他们一个有吸引力的模式进行研究ING顺-regulatory元素是控制基因表达胚胎发育过程中。在这里,我描述了开创性方法的适应性开发25年前在玻璃海鞘罗布斯塔为Ë ñ集体DNA Ë为lectroporation 体内吨ranscription额外物种小号属于三个属。类似的优化很可能会使电穿孔在其他海鞘物种中取得成功,其中可以对大量卵子进行体外受精。


[背景]海鞘是海洋无脊椎动物,是最接近的脊椎动物亲属。他们的快速,刻板,和外部的胚胎发育产生了蝌蚪状幼虫非常类似于其他脊索动物(脊椎动物和文昌鱼)。它们紧凑的基因组,加上操作基因表达和功能的简单有效方法的可用性,使海鞘成为功能基因组学的有趣模式生物(Lemaire,2011;Satoh,2014)。特别地,在引入质粒DNA到受精卵通过电击(电穿孔)使数百至数千瞬时转基因胚胎的产生,这促进了玻璃海鞘罗布斯塔大约在25年前为基准海鞘物种(CORBO等人, 1997) 。DNA电穿孔是一种广泛使用的方法为体内转录测定法(对于候选的活性的表征顺使用报告基因-regulatory元素),过表达/击倒,和实时成像。
海鞘是动物的不同群体的小号含有约3 ,000种是已被证明的有趣的系统,研究发展机制的演化,因为它们的基因组已被广泛重新排列,但其胚胎发育强烈保守(Dardaillon等人。2020年) 。特别是,DNA 电穿孔已应用于各种物种(Roure等人,2014 年;Stolfi等人,2014 年;Colgan等人,2019 年;Coulcher等人,2020 年)。在这里,我提供了一个详细的协议来执行在电穿孔4种:Phallusia mammillata ,Phallusia fumigata ,Ascidia mentula ,和Molgula appendiculata (图1) ,其覆盖近400万ÿ耳朵进化。

关键字:海鞘, 被囊动物, 电穿孔, 转基因技术, DNA顺式, 海鞘属, 海鞘, 莫古拉



材料和试剂
 
一次性手术刀
300 -和 120 - µm 自制筛子
Ñ OTE:剪切50 -在约4毫升的塑料管-从开口5厘米。用铝箔盖住加热板。使用加热板将一块尼龙网(Sefar Nitex,目录号:03-300/51 [ 300 - µm 开口] 、03-120/49 [ 120 - µm 开口] )与切割管融合。
15 - ml 玻璃离心管(Dutscher,目录号:092305)
15 - ml塑料锥形管(例如,Sarstedt,目录号:62.554.502)
60 - mm 塑料培养皿(例如,Sarstedt,目录号:82.1194.500)
92 - mm 塑料培养皿(例如,Sarstedt,目录号:82.1473.001)
60 -毫米和92 -毫米琼脂糖-涂覆的培养皿中。或者,可以像Sardet等人一样使用明胶涂层菜肴 (GF) 。( 2011)
Ñ OTE:熔体的在海水中的琼脂糖(每100ml海水1g)的。填充92 -毫米培养皿与热溶液。将琼脂糖溶液倒入下一道菜中,制作一层薄薄的琼脂糖层;重复,直到你有足够的菜肴(需要2种每电菜肴,加上dechorionation,施肥一打,洗荷兰国际集团)。
6孔板
注意:鸡蛋往往会粘在新的塑料材料上。为了避免这种情况,重新我们ē相同板; s意味着用自来水清洗并风干。
手套(假阴茎血是一种强大的污渍!)
玻璃巴斯德吸管
注:浸泡其中自来水,以免粘在的胚胎小号。使用打火机平滑开口。准备一些与一个较大的开口(2 -使用用于在Phallusia鸡蛋收集金刚石3毫米)。
100毫升玻璃烧杯
水平旋转振动器(例如,VWR,目录号:444-2900)
1.5毫升微管
低-结合1.5毫升(例如,VWR,目录号:525-0230)和2毫升(例如,VWR,目录号:525-0232)微管
4 mm 电穿孔比色皿(例如Dutscher,目录号:38191)
凹陷载玻片:4 mm(Dutscher,目录号:065230)和 1.5 mm(Dutscher,目录号:65227)的厚度
钨针(Roboz Surgical Instrument Co.,目录号:RS-6065)
成年海鞘是通过潜水、拖网或挖泥船从海洋中收集的,由欧洲研究基础设施 EMBRC 的法国节点提供(Station biologique de Roscoff:Phallusia mammillata和Ascidia mentula ;Station Marine de Banyuls-sur-mer:Phallusia mammillata ,Phallusia fumigata ,Ascidia mentula ,并Molgula appendiculata )
报告基因构建体的超螺旋环状质粒 DNA (20 - 100 µg)
氢氧化钠,2.5M
氢氧化钠,1M
1 M Tris,pH 9.5
0.96 M D-甘露醇溶液(VWR,目录号:25311.297)
TE(10 mM Tris-HCl,1 mM EDTA,pH 8.0)
25%戊二醛溶液(Sigma-Aldrich,目录号:G6257)
巯基乙酸钠(Sigma-Aldrich,目录号:T0632)
链霉蛋白酶(Sigma-Aldrich,目录号:P5147)
0.2 µm过滤天然海水或人造海水(BASWH:见配方)
Phallusia和Ascidia d回声溶液(见食谱)
Molgula dechorionation 解决方案(见食谱)
PBTw(见食谱)
10 × PBS(见食谱)
X-gal 染色溶液(见配方)
X-gal 原液(见配方)
 
设备
 
剪刀和镊子
温控室设置在 18°C
活胚胎操作的解剖范围(例如,蔡司的 Discovery V8)
方波电穿孔器(Harvard Apparatus,BTX ECM830,目录号:45-0052)
孵化器
解剖范围配备有一个数字照相机用于染色分析(例如,发现V20 + AxioCam型Erc从蔡司5S)
 
程序
 
的体内转录测定法进行根据这些连续的步骤:1)收集配子,2)蛋dechorionation,3)施肥,4)DNA电穿孔,5)胚胎固定和染色,和6)的数据收集和分析。前 4 个步骤因物种而异。在这里,我提出2个版本:一为P. mammillata ,P.熏蒸,和A. mentula (含有少量的变化之间的物种); 和M. appendiculata 之一。
 
 
图 1. 成年海鞘物种。(A)Phallusia mammillata (5 - 20cm长)。(B)Phallusia fumigata (5 - 20cm长)。(C)Ascidia mentula (4 - 10cm长)。(d)Molgula appendiculata (3 -长6厘米)。
 
配子收集
海鞘是雌雄同体的。其目的是同时收集鸡蛋和配子,分别,从每个个体。
假阴茎和海鞘
P. mammillata ,P.熏蒸,和A. mentula属于同一家庭,Phlebobranchia。通过解剖从性腺收集配子。为了避免不必要的自体受精,先收集卵子。所有物种都有透明的卵。
打开的通过之间切割用手术刀动物2个虹吸管(图2A)。阴茎外衣又厚又硬。
将外衣向两侧的动物底部切开。做ñ Ø牛逼切割过深,以免损坏动物。
用手指拉开束腰外衣。
将动物转移到其左侧的盘子中(您应该看到心脏在动物底部跳动,与虹吸管相对)。如果动物在右侧,您应该看到一个充满卵的大的、淡黄色(假阴茎,图 2C)或淡红色(海鞘,图 2B)的输卵管。
使输卵管小切口和收集的鸡蛋巴斯德吸管(图2D)。
Ñ OTE:在输卵管的卵是非常紧凑的并嵌入在克EL atinous物质。使用wide-孔巴斯德吸管的Phallusia ; 否则,它们通过移液管的小开口吸入可能会导致极度变形和卵子死亡。死蛋很容易被发现,因为它们变得不透明。
 
 
图 2. Phallusia和Ascidia 中的配子集合。(A)示出了示意性的连续的切口图(编号为红色虚线)以打开外衣(左:与两个虹吸管指向侧视图到左侧;右:侧面图与两个虹吸管朝向实验者指向)。(B) 解剖A. mentula个体,其中性腺暴露。(C) 解剖P. mammillata个体,其中输卵管清晰可见。(d)卵收集在P. mammillata使用宽-孔巴斯德吸移管。(E) P. mammillata蛋。(F) A. mentula蛋。比例尺:D 和 E 中的 100 µm。
 
转移的鸡蛋到一个装满海水的6孔板。Phallusia个体通常具有一个高数蛋(0.5 - 2毫升蛋)。Ascidia通常有较少的鸡蛋,但量能达到0.5 - 1毫升。
检查的蛋质量(透明而均匀卵细胞质,图2E和图2 ˚F )并传送它们到一个玻璃管中洗涤几次与海水。卵可以保持过夜,在14℃下,传播ö n中的陪替氏培养皿,没有大的降低在质量。
取出卵子后,您应该会看到输卵管下方的输精管(尖锐的白色导管)。使一个小的切口,并收集所述精子到1.5 -毫升管使用用于巴斯德吸管Phallusia或微量移与黄色尖端。避免收集海水、碎屑和血液。干燥的精子在 4°C 下可保持活跃数天。
 
莫古拉
与Phallusia和Ascidia 不同的是,微小输卵管中的卵很少(图 3D)。配子是第erefore由性腺收集人清扫。从性腺收集的卵母细胞代表卵子发生的所有阶段。该程序旨在收集主要是尚未成熟的完全发育的卵母细胞,如存在大的生发囊泡 (GV) 所证明的那样。幸运的是,这些卵母细胞成熟的自发在海水中,和胚泡破裂(GVBD)30内发生- 60分钟。
打开该用剪刀动物,从一个虹吸动物周围开始一路其他虹吸(图3A)(M. appendiculata都覆盖着碎片,贝壳,和石头; ü SE大剪刀和使周围这些障碍你的方式) .
拉开束腰外衣,拖动仍由虹吸管连接的动物(图 3B)。
用细剪刀将体壁从虹吸管剪到虹吸管,然后拉开动物(图 3C)。
在解剖范围下,用镊子在咽篮下找到性腺(动物的每一侧,图 3C)。您应该看到指向虹吸管的短性腺管和一侧被睾丸覆盖的大卵巢(图 3D)。
用剪刀剪下每个性腺,并将它们放在装满海水的 6 孔板的同一口井中(图 3E)。
 
 
图 3. Molgula appendiculata 中的配子集合。(A) 示意图说明切口 (红色虚线) 执行打开外衣 (左: 侧视图, 两个虹吸管指向顶部; 右: 侧视图)。(B) 去除束腰外衣(左侧)后的个人(右侧)。(C)剖开的具有性腺可见动物以在每一侧的中心。(D) 每个性腺由一个被睾丸包围的卵巢组成。入口示出了靠近-微小spermiduct(顶部)和输卵管(底部)的放大视图,两个开口指向右侧。其中性腺对(E)A 6孔板从4个个体已经被收集。(F) 在完全生长的卵母细胞自发成熟后获得受精卵 (比例尺: 50 µm)。
 
下的解剖范围,松开的从卵巢的卵母细胞使用镊子,和睾丸转移至培养皿填充有海水(60 -mm直径ETER )。您应该看到各种卵母细胞(从微小的透明卵母细胞到带有 GV 的大的、不透明的、完全生长的卵母细胞)。
传输所有的通过从一个个体(2个卵巢)卵母细胞中一个300放置在100μm筛毫升玻璃烧杯(性腺碎屑不会通过)。
传送内容的第烧杯到一个120微米筛,并与海水广泛洗到删除精子和卵母细胞小。
转移的卵母细胞于60毫米的琼脂糖包被的培养皿中。
等待30 - 60分钟,直到发生GVBD并产生受精的卵母细胞。
释放的从精子的那公顷的所有个人的睾丸VE在相同的培养皿中解离通过用镊子每个睾丸被收集(丢弃该测试ë š事后)。收集集中的精子溶液置于15 -毫升的塑料管中并保持在4℃直至使用。
 
卵脱层
保护绒毛膜小号卵子制成的内部卵黄膜的组成的细胞外物质和毛囊细胞的外泡沫层。为了执行电穿孔,有必要除去它; Ť他的使用巯基乙酸钠和链霉蛋白酶的混合物来实现的。每种化合物的浓度和脱膜作用的持续时间因物种而异。
注:去绒毛通常在受精前进行,裸卵仍可受精。对于 Molgula,由于该过程相当快,因此可以在受精后进行。
 
收集的鸡蛋在玻璃管中,并允许他们到沉在底部。
丢弃了多余的海水。
激活的通过提高pH dechorionation溶液(添加3滴(约100微升)(Phallusia / Ascidia ()或6滴(约200微升)Molgula )的2.5M的NaOH到7毫升dechorionation溶液使用巴斯德吸移管,并混合)。
继续装饰
我n以下的琼脂糖涂层培养皿为Phallusia / Ascidia ,通过将卵到dechorionation溶液(〜7毫升对于60 -毫米培养皿中并〜14毫升用于92 -毫米皿)。充分混合并以 ~70 rpm 的速度放置在水平振动筛上。
我Ñ一个玻璃管Molgula ,加入3 - 4毫升dechorionation溶液。通过上下移液充分混合。
定期检查范围内的除皱状态。Dechorionation需要30 - 45分钟(Phallusia / Ascidia )和7 - 15分钟(Molgula取决于)的蛋密度。
停止dechorionation当大部分鸡蛋都没有明确的的绒毛。Phallusia / Ascidia :通过旋转盘子在中心收集鸡蛋并将它们转移到15 ml 玻璃管中。将海水加入顶部并通过上下移液轻轻混合。Molgula :丢弃尽可能多dechorionation解决方案尽可能。将海水加入顶部并通过上下移液轻轻混合。
注:Dechorionation更难以用Molgula遵循:当毛囊细胞迅速消失,卵黄膜是比较难看到(看近LY ,改变的范围镜方向;测试单元的蛋的表面上的存在是一个很好的迹象是的鸡蛋没有完全dechorionated)。
洗广泛2 - 4次被允许荷兰国际集团的鸡蛋,以安定下来,更换的海水,并轻轻地用巴斯德吸管混合。碎片(绒毛膜和死卵)会漂浮,而裸卵会迅速沉入管底。裸鸡蛋易碎易爆;动作要轻柔,避免气泡。
您可以使用同一管继续下一步,或将鸡蛋收集在琼脂糖涂层的培养皿中以备后用。Dechorionated ,未受精卵可存储过夜,在4 - 14°与质量没有大的下降℃。
 
施肥
(Phallusia / Ascidia只)添加5μl的干精子到一个1.5 -含有1ml海水毫升微管中。
(可选)使用纯精子溶液通常可以有效受精;^ h H但是,要实现一个完整的受精率和高同步性,最好是通过提高pH值(在海洋,精子激活精子的解决方案是由海水的弱碱性pH值激活)。添加任一50微升1中号的Tris pH值9.5(Phallusia ),4微升1中号的NaOH(Ascidia ),或8微升1中号的NaOH(Molgula )到精子溶液。
注意:也可以通过将 1 ml 混合物与 20 µl 绒毛膜卵一起孵育 15 分钟来激活阴茎精子,如Sardet 等人所述。( 2011) 。
检查的通过的等分试样稀释50精子活力- 100 ×在海水和外表下在高放大倍率的解剖范围ING。精子应该疯狂地游动。
添加〜200微升(60 -毫米培养皿)或400 - 500微升(92 - mm皿或15 -毫升的玻璃管)精子溶液。
拌匀,麦荷兰国际集团的鸡蛋漂浮在介质中。你应该看到的精子游泳集中。
检查对发生的蛋变形小号受精,并应前几分钟内可见茨。
在10 - 15分钟后的施肥,进行到所述洗涤以除去精子,因为该协议使用大批量的精子。(施肥一个培养皿)通过旋动盘收集在中心处的鸡蛋并将它们转移到一个15 -使用巴斯德吸移管毫升的玻璃管中。允许卵到沉降到管底部,弃去大多数使用海水的一个巴斯德吸管,通过轻轻倒出加起来清洁海水顶端向下倾斜管的侧面; 由流感略有相似之处彻底悬浮鸡蛋^ h荷兰国际集团的海水用吸管。重复此洗涤1 - 3 × 。
注:胚胎没有他们的保护绒毛膜是脆弱的,一旦他们进来爆炸到与水/空气界面接触。小心谨慎;轻轻操纵它们,避免气泡。
 
DNA电穿孔
制备的DNA(20 -在50微升TE 100微克)。
加入 200 µl 0.96 M D-甘露醇并混合均匀。
收集的在玻璃试管底部受精卵。
标记用钢笔,在100上的低结合1.5μl的水平毫升管。
转移的受精卵进入的1.5 -毫升低-结合管。尝试将相同数量的鸡蛋添加到每个管中(每个要测试的构建体一个)。
用海水将体积调整到 100 µl 标记。
向每个管中加入 250 µl DNA/甘露醇溶液。
Ñ OTE:甘露醇是用来减少所述盐的浓度(并由此避免电弓ING电穿孔期间),同时保持所述容量渗透摩尔浓度在足够的水平。
转移的总混合TURE到电穿孔杯中(使用不同的巴斯德吸移管为电子ACH管,以避免混合DNA)。
将比色皿放入电穿孔器比色皿支架中。
应用 37 V ( Phallusia / Ascidia ) 或 20 V ( Molgula )的单个电脉冲32 ms。脉冲应该朝向第一细胞周期(但切割之前)的端部来进行:在50 - 60分钟后的受精。
Ñ OTE:电穿孔可以进行受精后任何时间和定时不影响的电穿孔效率; ħ H但是,受精卵是第一细胞周期的最后三分之一期间更坚固以电穿孔(而且显微注射)。
继续进行下一个构建体的电穿孔。
完成所有电穿孔后,在比色皿中加入一些海水,然后将鸡蛋转移到 92毫米琼脂糖涂层的培养皿中(一次电穿孔通常至少需要两个培养皿)。
注:共有4 - 6次电是常规做每施肥轮。根据经验,可以进行10 - 12 次电穿孔。
检查解剖范围下的菜。您应该看到完整的胚胎(因为它们接近第一个细胞周期的结尾,您应该看到与第一次卵裂的准备相对应的肌浆和/或变形)和爆炸卵的碎片。
注意:对于具有透明卵的物种(Phallusia/Ascidia),死卵变得不透明后很容易被发现。在大多数情况下,有一小部分卵子无法通过电穿孔存活下来;Ť他瑟对应于易碎或未受精的卵子。如果s其中你会发现没有幸存的胚胎,主要有两条方面的解释:在受精失败(未受精卵的电脉冲后,系统爆炸)或电压过高。尽量让两个实验控制小号:非dechorionated和非电穿孔胚胎,并dechorionated和非电穿孔胚胎。
转移的菜肴的培养箱设定在所需的温度。
注意:14 - 19°C 是所有物种的共同温度范围。它们也可以在较低的温度下很好地发育,但速度会显着降低。Phallusia/Ascidia 在高达 22°C 的温度下也能很好地发育。
冲洗海水用巴斯德吸管传播胚胎(他们有一个非常高的倾向,以彼此粘住,如果他们是太接近)。避免移动的自卵裂球的8细胞期之前菜肴非常松散地附着到彼此。
 
胚胎固定和染色
根据报告基因的不同,行动过程可能会有所不同。在这里,我描述了使用的LacZ作为报告基因(检测荷兰国际集团β使用发色底物X-gal的β-半乳糖苷酶活性)。
允许胚胎来发展,直到将所需的阶段。
旋转盘子以在中心收集胚胎。
收集的胚胎使用的玻璃巴斯德吸管,并将它们转移到一个2 -毫升低结合微管中。
允许胚胎来解决和丢弃的多余的海水。
用固定剂(海水中的 0.2% 戊二醛)填充管子,然后用手指旋转管子 30秒。
修复在室温下恰好30分钟(过定影将废除β半乳糖苷酶酶活性,太轻一个固定可导致胚胎崩解)。
取出一半的固定液并加入 1 ml PBTw。通过反转 2 - 3 × 进行混合。
一旦胚胎已经沉降d ,用 2 ml PBTw 替换溶液,倒置混合,等待 10 分钟。重复此洗涤1 × 。
漂洗用于在X-gal的5分钟染色溶液。
替换为 250 µl 含有 0.4 mg/ml X-gal 的染色溶液(将 X-gal 原液稀释100 × )。
孵育在37℃下(一个培养箱优选一个水浴,因为没有蒸发发生在管)。
不时检查蓝色染色(它会在几个小时内开始,具体取决于测试监管区域的强度)。
一旦染色充分(这可能需要几天时间,但这是一个永远不会产生任何背景的染色程序),在 PBTw 中洗涤 2 × 5分钟。可以在(Roure等人,2014 年;Coulcher等人,2020 年)中找到使用靶向细胞核的 β-半乳糖苷酶进行染色的典型例子。
后修复程序为在室温下或O / N在含有3.7%甲醛PBTw 1个小时,在4℃。
样品可以长期储存-项。
 
数据分析
 
数据采集
丢弃所述固定剂和洗涤2 ×在PBTw 10分钟。
传输所有的胚胎厚厚的抑郁症幻灯片。
将部分胚胎转移到薄的凹陷幻灯片上。
使用解剖范围和一个钨针,得分的数量染色的胚胎在靶组织,直到到达100 - 200刻痕胚胎或直至所有的在管胚胎已经取得(评分只有那些已发育正常胚胎)(罗亚等人,2014 年;Coulcher等人,2020 年)。
ñ OTES:
胚胎可以很容易地使用钨针被操纵,猫晶须,或睫毛。
根据目的,该方法不仅可以通过对染色胚胎的数量进行评分,还可以通过对每个胚胎的染色细胞数量进行评分来改进;ħ H但是,这可能是耗时的。
不要忘记,这种转基因方法会产生镶嵌转基因表达(每个细胞随机遗传了可变数量的质粒 DNA),并且每个胚胎都是一个独立的事件;因此,给定区域的活性是通过编译所有被分析胚胎中的表达来确定的。
以图像的代表胚胎。
 
顺式调节活性的评价和比较
该测定提供定性数据(即,给定基因组 DNA 片段的空间活动域),但染色胚胎的百分比也反映了调控区域的强度。
获得可以从实验变化根据实验得分的海鞘批次,质粒DNA的制备,并在电穿孔期间胚胎浓度; 吨HUS ,我们通常进行至少3次生物学重复(不同海鞘,不同天的实验)和计算平均得分和标准偏差。
Ñ OTE:长(4 - 10 KB)的基因组区域通常是强烈活性(染色是温育1个小时内可见,在37℃,用超过90%的胚胎染色),并且不显示实验之间太多变化。
通常,该测定的目标是定位必需的顺式调节元件;因此,需要比较各种结构的活性。显然,质粒骨架应该是相同的。我们也赞成比较的具有相同的实验过程中被电结构的结果。
 
食谱
 
Banyuls-sur-mer 人工海水 H EPES (BASWH)
BASWH是一个“地中海型”海水,具有以下特性:37.9克/升,1219 mOsmole,钠的重量克分子渗透压浓度盐度+ / K +比率的47 ,且pH的〜8.0。
为1升,米IX :
30.34 克氯化钠
0.83 克氯化钾
1.47 g CaCl 2 · 2H 2 O
4.98 克 MgCl 2 · 6H 2 O
6.29 g MgSO 4 · 7H 2 O在 MilliQ 水中
调整到 1 升
在这一步,溶液可以在室温下储存数月。
使用前,加入 0.18 g NaHCO 3和 5 ml HEPES 1 M pH 8.0,并滤液 (0.2 µm)。可以被存储为1 -在4℃下2个月。
假阴茎和海鞘脱膜液
1%巯基乙酸钠
0.05% 链霉蛋白酶在海水中
中号IX中的巯基乙酸通过摇动,加链霉顶部海水-没有混合-并保持在4℃下一个使用前几个小时。
在 4°C 下可使用 1 - 2 周。
使用前将等分试样置于室温。
Molgula dechorionation 解决方案
0.5% 巯基乙酸钠
0.1% 链霉蛋白酶在海水中
中号IX中的巯基乙酸通过摇动,加链霉顶部海水-没有混合-并保持在4℃下一个使用前几个小时。
在 4°C 下可使用 1 - 2 周。
使用前将等分试样置于室温。
PBTw
PBS 1 ×含 0.1% Tween-20
PBS 10 ×
对于 1 L,添加:
80.0 克氯化钠
26.8 克 Na 2 HPO 4 · 7H 2 O
2.0 克氯化钾
2.4 克 KH 2 PO 4
用 NaOH 将 pH 值调至 7.4
乙环至1L ,并高压灭菌
X-gal染色液
1 mM 氯化镁l 2
3 mM K 4 Fe(CN) 6
PBTw 中的3 mM K 3 Fe(CN) 6
X-gal 原液
40 mg/ml X-gal 在二甲基甲酰胺中
 
致谢
 
该协议用于Coulcher等人的研究“在不同的海鞘胚胎中保护周围神经系统形成机制” 。,于 2020 年在 eLife 上发表(doi:10.7554/eLife.59157)。由于 G. Diaz(法国 Port-Vendres 的专业渔民)和 Banyuls-sur-mer 和 Roscoff 海洋站的工作人员(潜水员和船员)的知识和慷慨,提供成年海鞘使这项工作成为可能(欧洲研究基础设施 EMBRC 的法国节点)。我要感谢我小组的成员。这项工作是由法国国家科学研究中心和索邦Üniversite电,并通过从ANR(ANR-11-JSV2-007和ANR-17-CE13-0027),国家科学研究中心(DBM2020与InSb)具体的资金支持,以及欧洲项目组装加( H2020-INFRAIA-1-2016-2017;授权号 730984)。
 
利益争夺
 
有没有利益或竞争利益冲突小号。
 
参考
 
Colgan, W., Leanza, A., Hwang, A., DeBiasse, MB, Llosa, I., Rodrigues, D., Adhikari, H., Barreto Corona, G., Bock, S., Carillo-Perez, A ., Currie, M., Darkoa-Larbi, S., Dellal, D., Gutow, H., Hokama, P., Kibby, E., Linhart, N., Moody, S., Naganuma, A., Nguyen , D., Stanton, R., Stark, S., Tumey, C., Velleca, A., Ryan, JF 和 Davidson, B. (2019)。被囊类心咽基因调控元件中不同水平的漂移。进化论 10:24 。
Corbo, JC, Levine, M. 和 Zeller, RW (1997)。来自海鞘的 Brachyury 启动子区域的脊索特异性增强子的表征。发展124(3):589-602。              
Coulcher, JF, Roure, A., Chowdhury, R., Robert, M., Lescat, L., Bouin, A., Carvajal Cadavid, J., Nishida, H. 和 Darras, S. (2020)。不同海鞘胚胎周围神经系统形成机制的保护。Elife 9 :e59157 。
Dardaillon, J., Dauga, D., Simion, P., Faure, E., Onuma, TA, DeBiasse, MB, Louis, A., Nitta, KR, Naville, M., Besnardeau, L., Reeves, W ., Wang, K., Fagotto, M., Gueroult-Bellone, M., Fujiwara, S., Dumollard, R., Veeman, M., Volff, JN, Roest Crollius, H., Douzery, E., Ryan , JF, Davidson, B., Nishida, H., Dantec, C. 和 Lemaire, P. (2020)。ANISEED 2019:对更多被囊类动物的基因数据进行 4D 探索。核酸 Res 48(D1):D668-D675。              
Lemaire, P. (2011)。发育生物学的进化十字路口:被囊类动物。发展138(11):2143-2152。              
Roure, A.、Lemaire, P. 和 Darras, S. (2014)。海鞘后神经发育的 otx/nodal 调节特征。PLoS 基因10(8):e1004548。
Sardet, C., McDougall, A., Yasuo, H., Chenevert, J., Pruliere, G., Dumollard, R., Hudson, C., Hebras, C., Le Nguyen, N. 和 Paix, A. (2011)。海鞘胚胎学方法:滨海自由城协议。方法 Mol Biol 770:365-400。
Satoh, N. (2014)。海鞘发育基因组学。新泽西州霍博肯,John Wiley & Sons, Inc.
Stolfi, A., Lowe, EK, Racioppi, C., Ristoratore, F., Brown, CT, Swalla, BJ 和 Christiaen, L. (2014)。不同的机制调节远亲海鞘中保守的心咽发育和基因表达。Elife 3:e03728。
 
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Copyright Darras. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Darras, S. (2021). En masse DNA Electroporation for in vivo Transcriptional Assay in Ascidian Embryos. Bio-protocol 11(18): e4160. DOI: 10.21769/BioProtoc.4160.
  2. Coulcher, J. F., Roure, A., Chowdhury, R., Robert, M., Lescat, L., Bouin, A., Carvajal Cadavid, J., Nishida, H. and Darras, S. (2020). Conservation of peripheral nervous system formation mechanisms in divergent ascidian embryos. Elife 9: e59157.
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