Method Article

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

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DOI:

10.3791/54173

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August 8th, 2016

 , 
1Department of Molecular Biology, University of Wyoming

In This Article

Summary

Mechanisms of cellular and intra-cellular scaling remain elusive. The use of Xenopus embryo extracts has become increasingly common to elucidate mechanisms of organelle size regulation. This method describes embryo extract preparation and a novel nuclear scaling assay through which mechanisms of nuclear size regulation can be identified.

Abstract

A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally scales with the size of the cell, notably during embryogenesis when dramatic reductions in both cell and nuclear sizes occur. Mechanisms of nuclear size regulation are largely unknown and may be relevant to cancer where altered nuclear size is a key diagnostic and prognostic parameter. In vivo approaches to identifying nuclear size regulators are complicated by the essential and complex nature of nuclear function. The in vitro approach described here to study nuclear size control takes advantage of the normal reductions in nuclear size that occur during Xenopus laevis development. First, nuclei are assembled in X. laevis egg extract. Then, these nuclei are isolated and resuspended in cytoplasm from late stage embryos. After a 30 - 90 min incubation period, nuclear surface area decreases by 20 - 60%, providing a useful assay to identify cytoplasmic components present in late stage embryos that contribute to developmental nuclear size scaling. A major advantage of this approach is the relative facility with which the egg and embryo extracts can be biochemically manipulated, allowing for the identification of novel proteins and activities that regulate nuclear size. As with any in vitro approach, validation of results in an in vivo system is important, and microinjection of X. laevis embryos is particularly appropriate for these studies.

Introduction

The sizes of cellular organelles typically scale with the size of the cell, and this has been perhaps best documented for the scaling of nuclear size with cell size1-10. This is particularly true during embryogenesis and cell differentiation, when dramatic reductions in both cell and nuclear size are often observed11,12. Furthermore, altered nuclear size is a key parameter in cancer diagnosis and prognosis13-17. Mechanisms that contribute to nuclear size regulation are largely unknown, in part due to the complexity and essential nature of nuclear structure and function. The method described here was developed as an in vitro assay for nuclear size scaling that is amenable to biochemical manipulation and elucidation of mechanisms of nuclear size regulation.

Xenopus laevis egg extract is a well-established system to recapitulate and study complex cellular processes in an in vitro context. These extracts have revealed new fundamental information about several cellular processes including the assembly and function of the mitotic spindle, endoplasmic reticulum, and nucleus18-22. One key advantage to the extract system is that X. laevis egg extracts represent a nearly undiluted cytoplasm whose composition can be easily altered, for instance through addition of recombinant proteins or immunodepletion. Furthermore, one is able to manipulate essential processes by employing treatments that might otherwise be lethal in an in vivo context. Modifications of the egg extract procedure allow for isolation of extracts from X. laevis embryos rather than eggs, and these embryo extracts are equally amenable to biochemical manipulation23. During X. laevis development, the single-cell fertilized embryo (~1 mm diameter) undergoes a series of twelve rapid cell divisions (stages 1 - 8) to generate several thousand 50 µm diameter and smaller cells, reaching a developmental stage termed the midblastula transition (MBT) or stage 824-26. The MBT is characterized by the onset of zygotic transcription, cell migration, asynchronous cell divisions, acquisition of gap phases, and establishment of nuclear steady-state sizes rather than continual nuclear expansion as in the pre-MBT embryo. From stage 4 to gastrulation (stages 10.5 - 12), the volume of individual nuclei decreases by more than 10-fold11.

Here, the goal is to identify mechanisms responsible for these reductions in nuclear size during developmental progression. The approach is to first assemble nuclei in X. laevis egg extract and to isolate those nuclei from the egg cytoplasm/extract. These nuclei are then resuspended in cytoplasm from late gastrula stage embryos. After an incubation period, the nuclei from egg extract become smaller in late stage embryo extract. We reasoned that this would be a useful assay for identifying cytoplasmic components present in late stage embryos that contribute to developmental nuclear size scaling. Using this assay, coupled with in vivo validation, we demonstrated that protein kinase C (PKC) contributes to developmental reductions in nuclear size in X. laevis23.

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Protocol

All Xenopus procedures and studies were conducted in compliance with the NRC Guide for the Care and Use of Laboratory Animals 8th edition. Protocols were approved by the University of Wyoming Institutional Animal Care and Use Committee (Assurance # A-3216-01).

1. Preparation of X. laevis Egg Extract (adapted from27,28)

  1. Prime female X. laevis frogs a minimum of three days and a maximum of two weeks before egg collection with a single 100 IU injection of pregnant mare serum gonadotropin (PMSG).
  2. One day before the experiment, inject primed frogs with 800 IU of human chorionic gonadotropin (HCG) and place at 16 °C in 1 L per frog of 1/3x Marc's modified ringers (MMR). See Table 1 for all buffer compositions.
    ​Note: Some frogs will not lay eggs or will lay poor quality eggs. Typically 2 - 3 frogs are injected to ensure at least one frog will lay sufficient numbers of good quality eggs. The average frog will lay enough eggs to produce at least 1 ml of egg extract.
  3. Prepare 1 L 1/3x MMR with cold distilled deionized water (ddH2O), 500 ml buffer B, 1 L buffer C, 500 ml buffer D, and 100 ml buffer E.
  4. Transfer laid eggs to a glass crystallizing dish (150 x 75 mm). Using a plastic Pasteur pipet with the tip cut off, farm out white puffy activated eggs or lysed eggs. Only retain eggs with distinct and equal dark animal poles and white vegetal poles.
  5. Prepare 13 x 51 mm centrifuge tubes with 1 ml of Buffer E supplemented with 355 µM cytochalasin D added just prior to use in step 1.7.
  6. Dejelly and wash the eggs.
    1. Wash eggs briefly with cold 1/3x MMR in an appropriately sized beaker, changing the buffer 3 - 4 times.
    2. Remove the jelly coats from the eggs by incubation with Buffer B. This will take anywhere from 3 - 10 min. As the cloudy jelly coats are released from the eggs, change the buffer.
      ​Note: Dejellying is complete when the eggs appear tightly packed in the corner of the dish when tilted and vegetal poles are oriented downwards. Eggs are much more fragile after dejellying, so do not incubate eggs with Buffer B for longer than necessary and subsequent washes need to be performed very carefully.
    3. Wash eggs briefly with 3 - 4 changes of Buffer C.
    4. Wash eggs briefly with 2 changes of Buffer D.
    5. Wash eggs briefly with 2 changes of Buffer E.
  7. Fill centrifuge tubes with eggs using a wide bore glass pipette or plastic dropper. Aspirate off excess buffer. To pack the eggs and remove as much buffer as possible, centrifuge in swinging bucket rotor at 212 x g for 60 sec and then at 478 x g for 30 sec. Aspirate off excess buffer.
    ​Note: It is important to remove as much excess buffer as possible at this step to ensure the egg extract is minimally diluted.
  8. In a swinging bucket rotor, ultracentrifuge under vacuum for 15 min at 12,000 x g and 4 °C to crush eggs open and separate into different layers, with the cytoplasm being the amber-colored layer in the middle. Remove centrifuge tubes and place immediately on ice.
  9. Collect the extract.
    1. Using an 18 gauge needle and syringe, puncture the centrifuge tube just above the dark pigmented layer at the bottom of the tube and remove the middle amber-colored cytoplasmic layer. Do not collect any of the top lipid layer. Collect the cytoplasm in an appropriately sized tube.
      Note: Generally, 1 frog produces at least 1 ml of extract.
    2. Supplement cytoplasm with 1/1,000 the volume of 19.7 mM cytochalasin D, 1/1,000 the volume of LPC stock, and 1/50 the volume of 50x energy mix. Gently invert the tube to mix. Do not excessively pipet the extract.
    3. Keep the extract on ice and use it within 3 hr of preparation for best results.

2. Preparation of Demembranated X. laevis Sperm (adapted from29)

Note: The procedure volumes presented here are for up to 8 testes.

  1. Remove 0.05% benzocaine (10% benzocaine stock prepared in ethanol and diluted to 0.05% in frog tank water) from 4 °C and warm to room temperature. Anesthetize and sacrifice male X. laevis frogs in benzocaine solution at room temperature for 20 - 30 min. Ensure death by absence of heartbeat by examining and feeling the chest area, by lack of response to mechanical stimulation, and/or by decapitation.
  2. Make a U-shaped incision along the abdomen. Remove the tubulated mass of yellow fat bodies. At the top of the kidneys, locate the testes, which are oval-shaped pale pink masses about 1 cm in length29. Excise the testes and roll them on blotting paper to remove blood and other tissue.
  3. Wash testes in Buffer T. Use a tight fitted pestle to mince testes with 1 ml of Buffer T in a 1.5 ml tube until homogenous (10 strokes or more). Centrifuge 5 - 10 sec in a mini centrifuge and collect the supernatant, removing large pieces of tissue. Repeat the centrifugation once more and collect the supernatant.
  4. Transfer the sperm-containing solution to a 15 ml plastic tube. Increase the volume to a total of 2 ml with Buffer T. Centrifuge at 1,411 x g for 10 min at 16 °C to pellet the sperm.
  5. Remove the supernatant and resuspend the pellet in 500 µl of Buffer T. Centrifuge at 1,411 x g for 10 min at 16 °C. Repeat this step once or twice as needed to clean the pellet of somatic and red blood cells.
  6. Resuspend the pellet in 100 µl Buffer T and 300 µl Buffer S. Incubate at room temperature for 5 min.
    Note: Buffer S contains lysolecithin, which is responsible for demembranation.
  7. Add three volumes of Buffer R (prepared fresh before use). Invert tube exactly once. Centrifuge at 1,411 x g for 15 min at 16 °C.
  8. Resuspend the pellet in 400 µl Buffer R and then add an additional 2 ml of Buffer R. Centrifuge at 1,411 x g for 15 min at 16 °C.
  9. Resuspend the pellet in 50 µl Buffer T.
  10. Dilute 1 µl demembranated sperm nuclei with 9 µl Buffer T. Apply this dilution to a hemocytometer. Count the number of thin S-shaped sperm nuclei in one large 4 x 4 grid, and multiply that number by 100 to obtain the concentration of the stock in sperm nuclei per µl.
  11. Dilute the stock to 100,000 sperm nuclei per µl with Buffer T, resulting in a 200x stock. Prepare 3 - 5 µl aliquots. Flash freeze in liquid nitrogen and store at -80 °C.

3. Nuclear Assembly

  1. Supplement 100 µl of egg extract with 1.5 µl 35.5 mM cycloheximide (final concentration ~ 0.5 mM), 6 µl 20x calcium stock solution (final concentration ~ 0.6 mM), and 0.7 µl 200x sperm (final concentration ~ 700 sperm nuclei/µl). Scale the reaction volume up or down as necessary. Invert or gently tap to mix.
    ​Note: Extract cycled from metaphase into interphase provides a more robust and reliable platform for nuclear assembly.
  2. Incubate in a water bath at 16 - 20 °C for 90 min. Mix by gently tapping every 15 min to ensure nuclei remain suspended in the extract.
  3. Monitor progress of nuclear assembly at 45 min by preparing a squash as follows.
    1. Pipet 2 µl of extract onto a glass slide and add 2 µl nucleus fix.
    2. Overlay with a 22 mm2 coverslip. Image on an epifluorescence microscope using water, oil, or air 20 - 100X objectives and a DAPI filter.
      ​Note: Nuclei can be visualized by bright-field imaging, but are much easier to visualize by fluorescence.
    3. Use nuclei immediately or flash freeze aliquots in liquid nitrogen and store at -80 °C.
      Note: Adding 4% glycerol to the nuclei prior to freezing can help to maintain their integrity. Frozen nuclei are generally still viable and capable of nuclear import, however the freezing process can lead to disrupted or more structurally fragile nuclei. Nuclei up to a year after freezing can be used.

4. Preparation of X. laevis Embryo Extract

  1. Induce female X. laevis frogs to lay eggs as described in 1.1 and 1.2.
  2. Isolate testes from male X. laevis frogs as described in 2.2. Submerge testes in L15 media supplemented with 50 IU/ml of penicillin and streptomycin in a glass 35 x 10 mm Petri dish. Store at 4 °C for up to two weeks.
  3. Collect and fertilize eggs.
    1. Collect freshly laid eggs by holding frogs over a glass reusable Petri dish and promote egg laying by applying gentle pressure to the lower back near the cloaca. Squeezing the frog too hard can result in injury, leading to a bloated abdomen and requiring euthanasia29.
    2. Prop the dish at an approximate 45° angle, allow eggs to collect in the edge of the dish, remove all excess buffer, and add enough 1/3x MMR to barely cover the eggs. Fertilize eggs within 15 min of collection.
    3. Use a tightly fitted pestle to macerate and homogenize 1/4 of a testis in a 1.5 ml tube with 500 µl of high salt Modified Barth's Saline (High Salt MBS). Add macerated testes to the eggs and allow for fertilization to occur at room temperature for 15 - 20 min, then flood eggs with 1/3x MMR.
      ​Note: Effectiveness of testes decreases with storage time, therefore more testes tissue may be required for successful fertilization when using testes that have been stored for more than one week.
    4. Confirm fertilization by checking for contraction of the dark pigmented animal pole and by rotation of the embryos with their animal poles facing up, usually about 20 - 30 min after addition of crushed testes. Expect the first cell cleavage to occur within 90 min.
    5. Using a wide bore glass pipette diligently remove dead (white and puffy or uneven distribution of yolk and pigment), lysed, or unfertilized eggs (i.e., not cleaved), as they will rapidly induce death in neighboring embryos.
    6. Anywhere from 1 - 2.5 hr post-fertilization, dejelly embryos in 2 - 3% cysteine dissolved in 1/3 x MMR and adjusted to pH 7.9 with 10 N KOH. Perform two buffer changes that are each 3 - 4 min. Thoroughly wash the embryos with 6 - 10 brief changes of 1/3x MMR to remove all traces of cysteine.
      Note: Dejellied embryos have a vitelline membrane and are not as fragile as dejellied eggs.
  4. Using a wide bore glass pipette, transfer healthy fertilized (i.e., cleaved) embryos to a Petri dish containing fresh 1/3x MMR and allow them to develop to the desired stage at RT or, if slower development is preferred, 16 °C. Continue to remove dead or lysed embryos indicated by lack of first division or white puffy appearance.
  5. Verify the stage of the embryos by checking for nearly complete closure of the blastopore and by comparing to drawings of staged embryos available in reference materials24.
    Note: Embryos cultured at 16 °C will reach stage 11.5 - 12 in approximately 12 hr.
  6. Incubate stage 11.5 - 12 embryos in fresh 1/3x MMR supplemented with 0.5 mM cycloheximide for 1 hr at RT, to arrest embryos in late interphase.
  7. Transfer with wide bore glass or plastic pipette a minimum of 15 (preferably 30 or more) interphase-arrested embryos to a microcentrifuge tube.
    Note: 15 embryos are sufficient to produce approximately 20 µl of extract. Scale up as needed.
  8. Add 1 ml egg lysis buffer (ELB) supplemented with 1 µl of LPC stock. Gently invert to wash embryos, allow embryos to fall to the bottom of tube, and remove the buffer. Repeat this wash step two more times.
  9. Resuspend embryos in 500 µl of ELB plus 0.5 µl of LPC stock, 5 µl of 19.7 mM cycloheximide, and 5 µl of 35.5 mM cytochalasin D (to inhibit actin polymerization). Centrifuge at 200 x g for 1 min in a tabletop microcentrifuge.
  10. Remove excess buffer and use a pestle to thoroughly crush the embryos. Centrifuge in a swinging bucket rotor for 10 min at 10,000 x g and 16 °C.
  11. Puncture the lipid layer from the top with a 200 µl pipet tip. With a clean 200 µl pipet tip, remove the middle cytoplasmic amber-colored layer to an appropriate sized tube.
  12. Supplement embryo extract with 1/50 the volume of 50x energy mix (to provide an ATP regeneration system), 1/100 the volume of 35.5 mM cyclohexamide, 1/500 the volume of 19.7 mM cytochalasin D, and 1/1,000 the volume of LPC stock.
  13. Prepare a squash as described in 3.3 to visualize endogenous embryonic nuclei in the extract.
    Note: Embryo extract can be frozen with the endogenous nuclei at this point. Aliquot to reduce freeze/thaw cycles and store at -80 °C.
  14. To remove nuclei from the embryo extract, dilute the extract with an equal volume of ELB containing 1/50 the volume of 50x energy mix, 1/100 the volume of 35.5 mM cyclohexamide, 1/500 the volume of 19.7 mM cytochalasin D, and 1/1,000 the volume of LPC stock. Centrifuge in a swinging bucket rotor at 17,000 x g for 15 min at 16 °C.
  15. Collect the supernatant being careful to avoid any remaining lipid at the top. Do not disturb the pelleted nuclei at the bottom of the tube. Prepare a squash as described in 3.3 to ensure that most nuclei have been removed. Use the extract fresh or aliquot and store at -80 °C.
  16. To heat inactivate embryo extract for control experiments, heat 30 - 100 µl aliquots of extract at 56 °C for 30 min using a thermal cycler. Allow heat inactivated extract to return to room temperature prior to use.

5. Nuclear Shrinking Assay and Immunofluorescence

  1. Isolate nuclei assembled in egg extract by diluting 25 - 150 µl of pre-assembled nuclei in 1 ml ELB in a 1.5 ml tube. Centrifuge at 1,600 x g for 3 min at 4 °C in a tabletop microcentrifuge. Remove buffer, being careful not to disturb the fragile pellet of nuclei at the bottom of the tube.
  2. Resuspend nuclei in a volume of embryo extract equal to the original volume of egg extract used in 5.1. Use ELB or heat inactivated extract for control reactions, as appropriate.
  3. Gently tap the tube to break up the pellet and resuspend the nuclei. Incubate at room temperature for 90 min, gently tapping the tube to mix every 15 - 30 min. Note: Most of the nuclear shrinking occurs within the first 30 min.
  4. Monitor progress of nuclear shrinking by preparing a squash.
    1. Pipet 2 µl of extract onto a glass slide and add 2 µl nucleus fix.
    2. Overlay with a 22 mm2 coverslip. Image on an epifluorescence microscope.
  5. Tap the tube to resuspend the nuclei just prior to adding 500 µl of fixative consisting of ELB, 15% glycerol, and 2.6% paraformaldehyde. Invert immediately, and place the tube on a rotator for 15 min at room temperature.
  6. Prepare spin down tubes by outfitting 15 ml round bottom glass tubes with round bottom plastic inserts (design and schematics available upon request). Add 5 ml of nuclear cushion buffer. Drop a 12 mm circular coverslip into the tube, being sure it lays flat on top of the plastic insert.
  7. Gently layer the solution containing fixed nuclei on top of the nuclear cushion using a wide-bore pipet tip. Centrifuge in a swinging bucket rotor for 15 min at 1,000 x g and 16 °C.
  8. Use an aspirator to remove all of the buffer and pull the plastic insert to the top of the tube. Remove the coverslip with a pair of fine forceps, being careful to note the side of the coverslip onto which the nuclei were spun. Post-fix in cold methanol (stored at -20° C) for 5 min at RT.
  9. Transfer the coverslip nucleus side up onto a sheet of plastic paraffin film lining a large plastic petri dish. Place wet disposable wipes along the side of the dish to prevent dehydration.
  10. Rehydrate nuclei on the coverslip with 500 µl of PBS-NP40 (1x PBS plus 0.1% NP40) and aspirate after 5 - 10 sec.
  11. Carefully layer 75 µl of PBS-3% Bovine Serum Albumin (BSA) onto the coverslip. Allow to block 1 hr at room temperature or overnight at 4 °C.
  12. Remove blocking solution, and incubate with primary antibody (e.g., mAb414 against the nuclear pore complex, 1:1,000) diluted in PBS-3% BSA for 1 hr at room temperature or overnight at 4 °C. Wash with five immediate changes of PBS-NP40.
  13. Incubate with secondary antibody diluted in PBS-3% BSA for 1 hr at RT. Wash with five immediate changes of PBS-NP40.
  14. Incubate with 10 µg/ml Hoechst diluted in PBS-3% BSA for 5 min at RT. Wash five times with PBS-NP40. Remove all excess buffer.
  15. Mount the coverslip onto a slide with 5 µl antifade mounting medium. Seal with clear nail polish. Image immediately using an epifluorescence microscope with 20 - 100x objectives or store at 4 °C for later imaging.
    Note: Perform quantification as previously described23.

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Results

Assembly of Nuclei in Egg Extract

The first steps of this protocol are to prepare X. laevis egg extract (Protocol 1) and demembranated sperm nuclei (Protocol 2). These reagents are then used to assemble nuclei de novo (Protocol 3). Figure 1 shows some representative data. Addition of calcium drives the meiotically arrested egg extract into interphase, and the cycloheximide keep...

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Discussion

Here is presented a novel method to study mechanisms of nuclear size regulation during X. laevis development. Developmental progression is associated with dramatic changes in cell physiology, metabolism, division rates, and migration, as well as alterations in the sizes of cells and intracellular structures. These varied processes are complex and essential, so it is difficult to study just one of these aspects of development in an in vivo setting. The X. laevis embryo extract and nuclear shrink...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

Members of the Levy and Gatlin labs as well as colleagues in the Department of Molecular Biology offered helpful advice and discussions. Rebecca Heald provided support in the early stages of developing this protocol. This work was supported by the NIH/NIGMS (R15GM106318) and the American Cancer Society (RSG-15-035-01-DDC).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Alexa Fluor 568 Donkey anti-mouse IgGMolecular ProbesA10037
ATP disodium saltSigma AldrichA2383
BenzocaineSigma AldrichE1501
Bovine Serum AlbuminSigma AldrichA3059
CaCl2Sigma AldrichC3306
CentrifugeBeckmanJ2-21M
Centrifuge rotorBeckmanJS 13.1
chymostatinSigma AldrichC7268
creatine phosphate disodiumCalbiochem2380
cycloheximideSigma AldrichC6255
cytochalasin DSigma AldrichC8273
disposable wipes (kimwipes)Sigma AldrichZ188956
L-cysteineSigma AldrichW326306
EGTASigma AldrichE4378
FormaldehydeSigma AldrichF8775
Glass crystallizing dish (150 x 75 mm)VWR89090-662
GlycerolMacron5094-16
HEPESSigma AldrichH4034
Hoechst - bisBenzimide H 33342 trihydrochlorideSigma AldrichB2261
HCG - Human Chorionic Gonadotropin Prospechor-250-c
L15 MediaSigma AldrichL4386
leupeptinSigma AldrichL2884
LysolecithinSigma AldrichL1381
mAb414Abcamab24609
MgCl2EMDMX0045-2
MgSO4Sigma AldrichM9397
MaltoseSigma AldrichM5885
NP40BDH56009
ParaformaldehydeElectron Microscopy Sciences15710
Penicillin + StreptomycinSigma AldrichPp0781
pepstatinSigma AldrichP5318
PIPESSigma AldrichP6757
Plastic paraffin film (parafilm)Sigma AldrichP7793
KClSigma AldrichP9541
KH2PO4Mallinckrodt70100
KOHBaker5 3140
PMSG - Pregnant Mare Serum GonadotropinProspechor-272-a
NaClSigma AldrichS3014
NaHCO3FisherBP328
Na2HPO4EMDSX0720-1
NaOHEMDSX0590
PestleThomas Scientific3411D56
Round bottom glass tubes, 15 mlCorex8441
Secondary antibody (Alexa Fluor 568 donkey anti-mouse IgG)ThermoFisherA10037
sucroseCalbiochem8550
thermal cyclerBio-RadT100
UltracentrifugeBeckmanL8-80M
Ultracentrifuge rotorBeckmanSW 50.1
Vectashield (anti-fade mounting medium)VectorH-1000

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Xenopus ExtractsNuclear Shrinking AssayEgg Extract PreparationEmbryo Extract IsolationCytoplasmic Extract ManipulationNuclear Assembly ProtocolCentrifugation StepsMicroscopy VisualizationEmbryo Microinjection

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