Zygotic Fluorescence Recovery After Photo-bleaching Analysis for Chromatin Looseness That Allows Full-term Development

This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the University of Yamanashi. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Yamanashi (Permit Number: A24-50). All surgeries were performed under tribromoethanol anesthesia, and all efforts were made to minimize suffering. An illustrated overview of the procedures and time-table are shown in Figure 1 and Table 1, respectively.

1. Preparation of Messenger RNA (In Vitro Transcription of eGFP-H2B mRNA)

1. Linearize 5 µg of template DNA pTOPO-eGFP-H2B^3,7 with 30 U of Not I in 50 µL at 37 °C overnight.
2. Collect 1.5 µL of digested DNA for confirmation whether completely digested by electrophoresis.
   1. Apply 1.5 µL and 3 – 5 µL of undigested DNA with loading dye to the well of 2% agarose gel, respectively, and subject to electrophoresis.
      NOTE: If completely digested, a single band (approx. 5 kb) is observed. Undigested DNA is used as a control, which appears at a different position.
3. Add 151.5 µL of ddH₂O and 200 µL of phenol/chloroform/isoamyl alcohol (25:24:1) to the remaining digested DNA.
   1. Mix vigorously by vortex.
   2. Centrifuge at the maximum speed for 15 min.
4. Recover the supernatant and then add 200 µL of chloroform.
   1. Mix vigorously by vortex.
   2. Centrifuge at maximum speed for 15 min.
5. Recover the supernatant and then add 20 µL of 3M sodium acetate and 1.5 µL of ethachinmate.
   1. Mix vigorously by vortex.
   2. Add 550 µL of 100% ethanol and then mix vigorously by vortex.
   3. Centrifuge at maximum speed for 15 min.
   4. Discard the supernatant and then wash the pellet with 70% ethanol.
   5. Dry the pellet by evaporation for 5 – 10 min.
6. Dissolve precipitated plasmid DNA in 8 µL of nuclease-free water.
7. Assess plasmid concentration (expected concentration is about 300 – 500 ng/µL) with nanodrop by following manufacturer's instruction.
8. Perform in vitro transcription for mRNA encoding eGFP-H2B with 1 µg of purified DNA as a template in 20 µL of total reaction volume by following the manufacturer's instructions.
   1. After in vitro transcription, add 36 μL of water then recover 1.5 µL from 56 µL of synthesized mRNA for analysis by electrophoresis (as without poly A).
9. Perform poly A tailing for synthesized mRNA in a 100 µL reaction volume by following the manufacturer's instructions.
10. Add 60 µL of lithium chloride precipitation solution to the poly A tailed mRNA of eGFP-H2B and then mix vigorously.
    1. Cool at -30 °C for 30 min.
    2. Centrifuge at maximum speed for 15 min.
    3. Discard the supernatant and then wash the pellet with 70% ethanol.
    4. Dry the pellet by evaporation for 5 – 10 min.
11. Dissolve the pellet with 20 µL of nuclease-free water by heating at 55 °C for 30 min.
12. Assess the concentration of the precipitated mRNA with nanodrop. Dilute to 500 ng/µL with nuclease-free water and store at -80 °C until use.
13. Confirm preferred mRNA production; subject 1 µL of mRNA with/without (obtained in 1.8.1 and 1.12 steps, respectively) poly A tail mixed with 1.5 µL of 0.1 mg/mL ethidium bromide and 3 µL of sample loading dye to the electrophoresis analysis. Applied volume was 5.5 µL.
    NOTE: If Poly A tailing was successful, the shifted band compared to the mRNA without poly A tailing should be observed (Figure 2A).

2. Preparation of Vasectomized Male Mice

1. Weigh the ICR male mice at 8 – 12 months using a weight scale.
2. Intraperitoneally inject male mice with 0.7 – 0.9 mL 1.2% tribromoethanol anesthesia.
   NOTE: The amount of anesthesia depends on the size of the mouse. For example, a mouse weighing 40 g is injected with 0.8 mL of tribromoethanol anesthesia.
3. Wet the berry with 70% ethanol.
4. Make a small cut (3 – 5 mm) on the berry and then make an incision on the muscle wall with the help of scissors.
   NOTE: Scissors and forceps should be cleaned with 70% ethanol before starting the surgery.
5. Grip a fat pad with forceps and gently pull it out. Then, the testis and vas deferens appear.
   NOTE: Vas deferens is a U-shaped tube and with a red blood vessel.
6. Tie off the vas deferens at two points with surgical sutures and then cut out the middle part between the tied points.
7. Gently push back the fat pad and testis into the berry.
   1. Repeat the surgery with the remaining testis.
8. Sew the muscle wall with two stitches with surgical suture.

3. IVF

1. Inject 8 – 10-week old female B6D2F1 (BDF1) mice intraperitoneally with 7.5 IU equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) at 46 – 50 h interval.
2. Prepare drops of 300 µL human tubal fluid (HTF)⁸ media for capacitation and insemination 1 day before IVF in a 30-mm dish, cover these drops with the mineral oil on the laboratory bench and then preincubate in an incubator overnight.
3. Sacrifice matured male ICR mice by cervical dislocation. Dissect cauda epididymis with a 27-G needle and collect spermatozoon. Culture them for capacitation in 300 µL of HTF media for 1 – 2 h at 38 °C.
4. 16 h after hCG injection, sacrifice super ovulated female mice by cervical dislocation. Transfer the oviduct ampulla into mineral oil next to HTF media and then draw out cumulus cells and oocytes complex from this oviduct ampulla by a 30-G needle into the preincubated insemination-HTF medium.
   NOTE: Some female mice do not often show ovulation despite super ovulation treatment. Enlarged oviduct ampulla is a sign of ovulation.
5. After capacitation, transfer 2 – 3 µL of the capacitated spermatozoon into insemination-HTF media in which the ovulated oocytes were collected.
6. 1 h after insemination, collect the fertilized zygotes and treat them with hyaluronidase at 100 µg/mL for 10 min in CZB⁹ media.
   NOTE: When insemination is performed properly, 1 h after insemination, the fertilized zygotes with the 2^nd polar body are observed. If less or low-quality sperm is used, only little zygotes with the 2^nd polar body are observed. Also, if many sperms are used for insemination, polyspermy occurs. Proper insemination leads to zygotes with male and female pronuclei. By using these criteria, it is possible to find whether insemination is done well or not.
   1. After washing in CZB media, subject the zygotes with a second polar body to microinjection with eGFP-H2B mRNA.

4. Preparation of Chamber and Microinjection Pipettes

1. Dilute RNA encoding eGFP-H2B using nuclease-free water to obtain a concentration of 250 ng/µL.
2. Prepare 2 – 3 drops of PVP-HTF and eGFP-H2B drops mRNA, and 5 – 6 drops of Hepes buffered CZB (H-CZB) drops on the 60-mm dish. Cover these drops with mineral oil.
   NOTE: These preparations can be performed on the laboratory bench. There is no requirement for using laminar airflow cabinet.
3. Pull borosilicate glass with a micropipette puller (e.g., P = 500, heat = 825, pull = 30, vel = 120, and time = 200)
4. After breaking the tip of the pipette, bend the pulled glass microinjection pipette close to the tip (−300 µm back) at around 30° using a microforge.

5. Microinjection

1. Turn off the plate heater on the stage of micromanipulator to prevent the killing of the zygotes with a piezo during mRNA injection.
2. Wash and coat the inside of injection needles in HEPES-buffered-HTF containing 10% PVP (PVP-HTF).
3. Collect the fertilized zygotes with a 2^nd polar body (Figure 2B) and transfer 20 – 25 zygotes onto the chamber of the microscope stage attached with a micromanipulator.
4. Fill the diluted eGFP-H2B mRNA into borosilicate glass capillaries from the tips.
5. Hold the zygote with a holding pipette and then break the zona pellucida and the cytosolic membrane with a piezo drive.
6. Inject about 10 pL of mRNA into the cytoplasm of the zygotes. Finish mRNA-injection within 10 min to prevent damage caused by longer culturing the zygotes outside the incubator.
   NOTE: The injection volume should be as large as the 2^nd polar body. If the amount of mRNA injected is too large, it is possible to cause the free eGFP-H2B fraction, which may affect the mobile fraction.
7. 10 min after microinjection, wash in at least 3 drops and then culture the injected zygotes in CZB at 38 °C until zFRAP analysis.

6. zFRAP Analysis

1. 1 h prior to zFRAP analysis, turn on the laser and hot plate attached to the stage of the confocal microscope. Set the temperature at 38 °C.
2. Put 6 – 10 µL of HEPES-buffered CZB media covered with mineral oil on the 60-mm glass bottom dish and warm on the heater until zygote collection. 
3. 8 – 12 h after insemination, collect 5-10 eGFP-H2B-expressing zygotes and transfer into 6 – 10 µL of pre-warmed HEPES-buffered CZB medium drop.
   NOTE: To avoid longer culturing outside incubator, 5 – 10 eGFP-H2B-expressing zygotes were used at each analysis. 5 to 10 zygotes can be analyzed within 1 h.
4. Set the bleaching and imaging conditions according to the manufacturer's instructions. The case a confocal laser scanning microscope (Table of Materials) is shown below:
   1. Use 60X oil lens (60X 60X/1.35 oil).
      NOTE: Lenses with higher magnification (higher numerical aperture) show higher sensitivity.
   2. Set scanning speed as 4.0 µs/Pixel and size as "512" on "Acquisition Setting" (Supplemental Figure 1).
   3. Increase Digital zoom to "5" (Supplemental Figure 1).
      NOTE: A higher zoom is required to recognize whether focus drift occurs or not.
   4. Open dye list (Supplemental Figure 1) and then chose "EGFP". Set observation time as 1.6 s (interval is set as "free run" setting) (Supplemental Figure 1)
      NOTE: More observation points may cause more detailed data, but it may also cause phototoxicity. In the zFRAP analysis, a 1.6 s observation time seems to be enough to detect the parental asymmetry of chromatin looseness.
   5. Set the number of pictures as 12 in total (Supplemental Figure 1).
   6. Start "Stimulus setting" panel and then click "Main" on UseScanner. Set scanning speed as 10.0 µs/Pixel. Click "Activation in Series" and then set PreActivation as "3 frames" and Activation Time as "5 sec" (Supplemental Figure 1).
   7. Click "Focus x 2" and set the focus and then "Stop".
   8. Set bleaching and imaging laser power (477 nm) at 110 and 15 µW, respectively, by adjusting "laser gage power" (Supplemental Figure 1).
      NOTE: Very strong bleaching may cause a larger bleached area, which causes difficulties for the quantitative analysis. For measurement of the laser power, use a power meter. It is important to confirm the laser power to avoid the effect of the time-related deterioration of the laser power.
   9. Click "Clip Rect" (Supplemental Figure 1) in Stimulus Setting panel. Set the region of interest (ROI; red; ROI #1B) as 40 x 40 pixels (7.6 µm²). Prepare a reference region (REF; green; ROI #2), and a background (BG; blue; ROI #3) using "copy ROI" and "paste ROI" (right-click button on the mouse).
      NOTE: Pixel size can be set through "ROI manager" which can be called by clicking the right button of the mouse when the cursor is on the ROI. Larger ROIs are thought to be better since they can standardize the dispersion of the zFRAP score. However, too large of an ROI cannot be used for this analysis because it makes it difficult to avoid the nucleolus precursor body (NPB). eGFP-H2B in this compartment was thought to be free from chromatin and showed remarkable higher mobility³. ROI and REF areas should be separated as much as possible. If these two regions are close, bleaching may also affect the region of REF.
   10. Click "Live" on the tool bar and then start "Live Plot" (Supplemental Figure 1).
       NOTE: Live plot indicates fluorescence signal intensity within each ROI and is used for adjusting laser power using HV. Note that if ROI is not selected, no intensity would be detected on Live plot panel.
   11. Determining the ROI position
       NOTE: ROI can be moved by dragging into the desired position in pronuclei. (1) Be sure to avoid the nucleolus, and (2) fit the entire ROI into the nucleoplasm. Do not contain the region outside the area of the nuclear membrane (cytoplasm) in ROI. The localization of eGFP-H2B is highly restricted into the nucleoplasm so that it is easy to find whether ROI contains cytoplasm or not by the fluorescence of eGFP-H2B. Male pronuclei tend to be larger than those of females, which are often localized near the 2^nd polar body. While using these criteria, judge the male or female pronuclei.
   12. Click "Focus x 2" (Supplemental Figure 1) and then adjust the HV power gage of the channel for EGFP into fluorescence intensity to around 2000 (fluorescence intensity can be seen in the "live plot" window).
5. Click "Time" and then "XY" (Supplemental Figure 1) to start the zFRAP analysis.
   NOTE: If "Time" button is suitably selected, "t" appears on the "XY" button.
   1. Click "Series Done" (Supplemental Figure 1), which appears after FRAP imaging near the "XY button" and then obtain FRAP-analyzed data.
   2. Save the file "2D View-Image XXXXX" as a preferred named file (oib. file format) by "save as" (Ctrl + Shift + S can be also used).
   3. Select ROI, REF, and BG and then click (Ctrl+A can be also used) "Series Analysis" in "2D View Image" window.
   4. Obtain row FRAP data and then click "save" button in the live plot window.
      NOTE: Row FRAP quantitative data is saved as a csv file.
6. For no zFRAP control, put some of the zygotes injected with mRNA on the drop, which is near to the rim of the glass bottom dishes to avoid the effect from fluorescence observation.

7. Data Calculation

1. While using the obtained quantitative data, make the graph of the "recovery curve" and "mobile fraction" by Excel as shown in the references^3,7 with some modifications (see below and supplemental excel file).
2. Calculate the relative intensity by subtracting the value of BG from those of ROI and REF in 12 pictures for each time point.
3. Divide the value of ROI by that of the REF. As a result, 12 standardized relative intensity scores at each time point can be obtained.
4. Calculate the average of the relative score for ROI in 3 images taken before photo bleaching.
5. Calculate the recovery rate. Divide the 12 obtained relative scores for ROI in the images taken at each time point (obtained at 7.1.2.) by the average of the relative score before photo bleaching (obtained at 7.1.3.). Draw recovery curve with using these scores (Figure 2E).
6. Calculate the bleaching rate.
   NOTE: Bleaching rate (%) = 100 – recovery rate of 4^th image.
7. Calculate the mobile fraction (MF) by using the formula as follows^10,11,12
   MF (%) = 12^th recovery rate (at the end point) – 4^th recovery rate/ bleaching rate × 100.

8. Embryo Transfer

1. Mate matured ICR females at 8 – 12 months with vasectomized male ICR mice the night before the embryo transfer by letting them be in the same cage overnight.
2. Collect the embryos that were zFRAP-analyzed and determined to develop to the two-cell stage.
3. Intraperitoneally inject female mice with 0.7 – 0.9 mL 1.2% tribromoethanol anesthesia or with 0.35 – 0.45 mL of 0.75/4/5mg/kg medetomidine/midazolam/butorphanol mixed agents prior to embryo transfer.
   NOTE: The volume of anesthesia is determined by the body weight of mice. Tribromoethanol: 0.2 mL/10 g of body weight; medetomidine/midazolam/butorphanol mixed agents: 0.1 mL/ 10 g of body weight.
   1. Transfer these two-cell stage embryos into the oviducts of pseudopregnant female ICR mice with a vaginal plug at 0.5 days post coitum (dpc).
   2. After the embryo transfer, put the female mice on the heater set at 37 °C until they wake up.
4. At 18.5 dpc, sacrifice the surrogate mother by cervical dislocation and recover the pups derived from zFRAP-analyzed zygotes by cesarean section. Some of the recovered pups were delivered to the foster mother and their body weights were assessed each week.