TITLE: Generating cloned sheep embryos by zona-free somatic cell transfer
AUTHORS: Zachariah Mclean, Sarah Appleby, Lisanne Fermin, Bjorn Oback

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol summarises zona-free sheep cloning by somatic cell transfer (SCT), adapted from a similar SCT method in cattle (</span><a href="https://doi.org/10.1089/153623003772032763" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1089/153623003772032763</span></a><span>). It describes the workflow that is required for reprogramming somatic cells into cloned blastocysts of high morphological quality, including two optional steps of embryo aggregation and vitrification. The resulting embryos can then be transferred into surrogate ewes for producing live cloned animals. Compared to conventional zona-intact procedures, this zona-free embryo reconstruction system increases throughput of cloned embryo production and ease of operation. </span></div></div>

[STEPS]
?. [Induce donor cell quiescence by serum starvation]
Induce donor cells into quiescence (or G0) by serum starvation
?. [In vitro maturation of oocytes]
Retrieve oocytes from slaughterhouse ovaries
Sheep (ovine) is a seasonally reproductive species and SCT experiments are best carried out during the extended breeding season (February-July) when ewes are in estrous in our geographic region (latitude ˜37°S, Waikato, New Zealand). During that time, we collected ovaries from local slaughterhouses. Experimental outcomes are more consistent with ovaries from mature ewes rather than lambs. In slaughterhouses, designated “lambs” during the above specified collection period range from prepubescent to 13-months old. By contrast, “two-tooth” sheep (identified by the two adult central incisors which appear at approximately 13 months of age) are considered adult. On average, expect to recover 2 good quality oocytes per mature ewe ovary.
?. [Induce donor cell quiescence by serum starvation]
Seed cells at approximately 2.5 x 104 cells/cm2 in duplicate wells (equivalent to 5x104 cells per well in a 4-well plate or 5 x103 cells in a 96-well plate). Proceed to serum starvation within 6-20 hours after seeding.
?. [Induce donor cell quiescence by serum starvation]
Wash cells once with phosphate-buffered saline (PBS) and add starvation medium DMEM/F12+0.5% FBS for 4-6 days. Do not change medium during the starvation period.
?. [In vitro maturation of oocytes]
Aspirate follicles that range in size between -with the aspiration module (see materials section) using negative pressure from a vacuum pump (~43 mmHg) into -of oocyte aspiration medium in a 15 mL tube.
2 mL
3 mL
In order to keep oocytes warm, change tubes after filling to approximately 7 mL and keep at  .
?. [In vitro maturation of oocytes]
Transfer the aspirated follicular content (the sediment containing oocytes) from the 15 mL tube to a 90 mm dish containing fresh, warmed aspiration medium. Search for oocytes using a stereomicroscope with warm stage at, selecting only cumulus-oocyte complexes (COCs) with intact layers of cumulus and relatively homogenous ooplasm.
38.5 °C
All dishes used throughout protocol are Petri dishes to prevent adherence of cells.Wash dishes throughout protocol are 35 mm in diameter.All microscopes had a warm stage of unless otherwise stated.
?. [In vitro maturation of oocytes]
Wash COCs serially through two dishes of H199+FBS, then once in B199+FBS. Transfer 10 COCs in B199+FBS into a drop of IVM medium in a 60 mm diameter dish overlaid with mineral oil (maximum of 12 drops per plate).
10 µl
40 µl
It is important that B199+FBS and IVM dishes containing COCs do not stay out of the gassed incubators for so long that the pH changes (as indicated by colour change from red to pink).
?. [Collection of mature oocytes and zona removal]
Collection of mature oocytes and zona removal
?. [Collection of mature oocytes and zona removal]
Move MII-arrested oocytes at 19 hours post-onset of maturation (hpm) out of IVM drops into a wash dish of H199+FBS using a pipette. Transfer maximally 200 oocytes in as little medium as possible into hyaluronidase (in H199) in a 1.5 ml tube.
500 µl
?. [Collection of mature oocytes and zona removal]
Screen oocytes for the presence of a polar body (which in sheep is often flattened against the zona pellucida).Using a finely pulled glass pipette, rotate oocytes until the polar body is observed. After all oocytes have been screened, re-check those that were initially without a polar body. If there is still no polar body present, discard.
All following manipulations are performed using a finely pulled glass pipette attached to a mouth piece unless otherwise stated.
?. [Collection of mature oocytes and zona removal]
Wash oocytes in H199+BSA and allow 5–15 min for them to recover to a normal spherical shape before enucleation.
Some zonae are more resistant to pronase and will not be fully removed with pronasing alone. In these situations:If there is a small break in the zona, the oocyte can be released with a pipette whose tip is slightly smaller than the diameter of an oocyte (~120 µm). Alternatively, suck the oocyte up and down the shank of a standard pulled glass pipette until it is released from the zona.If the zona has loosened but is still intact, pin it to the bottom of the dish with a glass pipette (works best if the tip is not fire polished) while gently applying some suction. This pulls the oocyte and tears the zona, making a hole and releasing the oocyte.
?. [Enucleation of zona-free oocytes]
Enucleate zona-free oocytes
Enucleation occurs between 20-22 hpm.
?. [In vitro maturation of oocytes]
Rinse the ovaries very briefly in 70% ethanol to sterilise, followed by a thorough rinsing in saline 2-3 times.
30 °C
?. [In vitro maturation of oocytes]
Mature oocytes overnight atin humidified 5% CO2 with atmospheric O2 concentration.
38.5 °C
?. [Collection of mature oocytes and zona removal]
Vortex COCs for (-) to disperse cumulus cells. Briefly centrifuge to collect oocytes and transfer back into H199+FBS. Rinse the inside of the hyaluronidase tube with H199+FBS to recover any remaining oocytes.
Centrifuge: 2000 33
Centrifuge: 2200 33
?. [Collection of mature oocytes and zona removal]
Remove the zona pellucida by placing the polar body containing oocytes into a drop of pronase solution for 30 s – 2 min. Approximately 50 oocytes can be processed at a time, collecting misshaped oocytes with dissolving zonae first and exposing more pronase-resistant oocytes for longer.
50 µl
Sheep zonae tend to flatten to the bottom of the dish, making the oocytes harder to collect. Inexperienced users should start with smaller numbers until confident at lifting flattened oocytes off the bottom of the dish. Alternatively, flattened oocytes can be pushed to the edge of the pronase drop where surface tension lifts them from the base of the plate.
?. [Enucleation of zona-free oocytes]
DNA stain oocytes in groups of ~40 for in Hoechst 33342 in H199+BSA+Caffeine ('stain plate').
?. [Enucleation of zona-free oocytes]
Observe the DNA-stained oocytes under the 20x objective, with the fluorescence lamp diaphragm reduced as much as possible. Locate the ‘bump’ on the oocyte surface overlying the metaphase plate under brightfield-transmitted light illumination, then confirm presence of the metaphase chromosomes by briefly exposing them to UV light.
?. [Enucleation of zona-free oocytes]
Using micromanipulation, aspirate the metaphase chromosome-spindle complex into an enucleation needle (see materials section) with a minimal volume of cytoplasm. Aspirated material may include the first polar body, if still present. Separate the aspirated material (karyoplast) from the enucleated oocyte (cytoplast) by lightly tapping on the stage to vibrate the needle. Alternatively, the enucleation needle can be pushed past a separation needle (see materials section) to pinch off the karyoplast. Confirm aspiration of the chromosomes by brief UV exposure. As long as you can maintain suction control, proceed to enucleate the next oocyte without expelling the previous karyoplast.
?. [Enucleation of zona-free oocytes]
Transfer batches of 40 cytoplasts into the 'cytoplast plate' (30 per drop) until all oocytes are enucleated.
?. [Cell preparation and attachment]
Cell preparation and attachment
Cell preparation can start around 20 minutes before finishing enucleation.
?. [Cell preparation and attachment]
Passage cells by washing with PBS, followed by trypsinisation. Inactivate the trypsin with warmed H199+0.5% FBS. Lift cells and centrifuge. After centrifugation, remove supernatant and resuspend at approximately 1 x 105 cells/mL in H199+0.5% FBS. Aliquot the cell suspension into drops under oil.
40 µl
?. [Cell preparation and attachment]
Distribute a few hundred cells across four drops of the 'lectin plate' with phytohemagglutinin (PHA-P). While cells settle to the bottom of the dish, transfer approximately 80 cytoplasts into the remaining drops (10 cytoplasts per drop) and incubate in the lectin solution for a few minutes before cell attachment.
Inexperienced users may start with 5 cytoplasts per drop, as you gain experience this can increase to 10-12 per drop. Note that moving the dish around will slowly bring the cytoplasts closer to together, resulting in unattended cytoplasts sticking to each other.
?. [Cell preparation and attachment]
Attach a cytoplast by dropping it on top of a cell, rolling it a few times to increase adherence, and then re-depositing the couplet back into a drop devoid of donor cells. Incubate couplets in lectin for at least 5 minutes.
All couplets should be kept separate to avoid them sticking together. If this happens, cytoplasts have to be manually separated, which often creates membrane protrusions that look like donor cells, complicating scoring of successful cell fusion later on.If more than one cell sticks to a cytoplast, or two cytoplasts stick together, immediately pipette them up and down within the lectin drop until they separate. If they are attached more firmly (due to longer incubation in lectin), transfer the couplet to a wash dish or drop under oil of H199+BSA+Caffeine and pipette up and down until they separate. Strongly attached extra cells can be blown against the drop/oil interface to help knock them off.
?. [Cell preparation and attachment]
After incubation in lectin, wash couplets through H199+BSA+Caffeine and then store in the 'couplet plate' with a maximum of 10 per drop to prevent cytoplast-cytoplast adhesion (couplets are still adherent after removal from lectin).
?. [Cell electrofusion (SCT)]
Cell electrofusion (SCT)
The actual SCT step occurs by electrofusion at approximately 22-24 hpm
?. [Cell electrofusion (SCT)]
Remove batches of 20-40 couplets from drops under oil and wash through a dish of H199+BSA+Caffeine, then wash through two dishes of fusion buffer (see materials).
It is critical to use fusion buffer without calcium ions to prevent premature cytoplast activation.
?. [Cell electrofusion (SCT)]
Prepare for electrofusion by attaching the chamber (see materials section) to a 90 mm dish with Blu-Tack and flooding it with fusion buffer. To reduce evaporation, fusion is performed at room temperature, not on a microscope warm stage. Use a pipette to remove any bubbles within the chamber and electrodes. Connect the electrodes to the fusion machine (see materials section).
0 Room temperature
?. [Cell electrofusion (SCT)]
Transfer 5-20 couplets to the electrofusion chamber. Couplets are automatically aligned by applying an alternating current (AC) field (83 V/cm) for 5–10 s. If any couplets do not automatically align, remove them from the chamber. Apply 2 x 10 µs, 2 kV/cm direct current (DC) pulses to the automatically aligned couplets. Manually align remaining couplets and apply the same DC pulses.
?. [Cell electrofusion (SCT)]
Return couplets to fusion buffer dish until all have been exposed to the DC fusion pulse, then wash through H199+BSA+Caffeine. Place the couplets in the 'post-fusion plate' (10 couplets per drop) for scoring cell fusion 10–30 minutes post-DC pulses.
?. [Chemical activation]
Chemical activation
Chemical activation is carried out 3–4 hours post-fusion to prolong the donor nucleus exposure to the MII cytoplast environment.
?. [Chemical activation]
Equilibrate the reconstructs by transferring them from their single drops in the ESOF-Ca plate to a dish of HSOF +1 mg/ml BSA at 5–30 minutes before activation.
?. [Chemical activation]
Transfer the reconstructs through series of three wash drops of 6-DMAP before placing individual reconstructs into single drops of 6-DMAP ('DMAP plate'). Incubate for 3–4 hours at in a humidified 5% CO2 incubator with atmospheric O2 concentration.
5 µl
38.5 °C
?. [In vitro embryo culture]
Embryo culture
The following embryo culture incubation steps are at 7% O2 in modular incubation chambers with 5% CO2 in N2. (Previous incubation was at atmospheric O2 concentrations.)
?. [In vitro embryo culture]
Following HSOF washes, transfer the zona-free embryos to the 'ESOF plate'. Wash embryos serially through two wash-drops. Single embryos are then placed in manually prepared micro-wells (dimples) to prevent the embryos from aggregating. Embryos are cultured in groups of 10 in and stored in 7% O2 in modular incubation chambers.
40 µl
20 µl
Micro-wells are made by using a blunt needle to push dimples in the plastic in the base of the Petri dish after drops have been made. Sterilise the blunt needle first with 70% ethanol and rinse in HSOF. Embryos can be successfully cultured in groups of 8-12, if necessary.
?. [In vitro embryo culture]
On day four of culture, replace three quarters of the medium () with warm, gassed LSOF and return to 7% O2 in modular incubation chamber.
15 µl
?. [Morula aggregation]
Morula aggregation
?. [Morula aggregation]
Select competent morulae with little fragmentation on day five of embryo culture. Compact morulae are the preferred stage for aggregation, but pre-compacting and compacting morulae can also develop into good quality aggregate blastocysts, albeit at lower efficiency.
Compact morulae often adhere to the micro-well surface, which helps in their identification during transfer into LSOF droplets. In some instances, morulae may also be found and aggregated on days four and six of culture.
?. [Morula aggregation]
Incubate paired morulae in disaggregation media (see materials section) for , wash in HSOF, and transfer todrops of LSOF containing one micro-well.
5 µl
?. [Morula aggregation]
Gently disaggregate the embryos by blowing up and down with a pipette smaller than the embryo (inner diameter: 50-70 µm) until they break into 2-3 fragments.
?. [Morula aggregation]
Re-aggregate the embryo fragments with a pipette approximately the diameter of the reconstituted embryo and place into a micro-well. Aggregated embryos are then incubated in the modular incubation chamber for 5–6 hours.
?. [Morula aggregation]
Remove each aggregated embryo from the micro-well after 5–6 hours, before blastulation occurs. Within this time, the pieces usually form a single compacted morula. Each individual aggregated embryo is then placed into a single LSOF drop for culture until day six or seven at 7% O2 in a modular incubation chamber. Grade blastocysts as in 8.5.
Blastocysts are removed at this stage since the trophoblast adheres tightly to the edges of the micro-well and often becomes damaged upon removal.
?. [Cryopreservation by vitrification]
Cryopreserve by vitrification
?. [Cryopreservation by vitrification]
Cryopreserve blastocysts using the CryoLogic Vitrification Method (CryoLogic, Australia), following an adapted protocol from our lab.
?. [Cryopreservation by vitrification]
Wash blastocysts twice through base medium (Embryo-hold (no BSA)+20% FBS), then incubate in a solution comprised of 7.5% (v/v) each of ethylene glycol and DMSO in base medium for .
?. [Cryopreservation by vitrification]
Transfer the embryo to a solution of base medium with the addition of Ficoll 70, sucrose and 15% (v/v) each of ethylene glycol and DMSO for for equilibration. Collect embryos in 2.5 µl and carefully deposit as a single droplet on the nylon hook of a CryoLogic fibreplug.
Embryos can be vitrified in groups of 1-4 per hook.
?. [Cryopreservation by vitrification]
Sheath the fibreplug in a cooled plastic cover and store in liquid N2 until day of embryo transfer.
?. [Cryopreservation by vitrification]
On the morning of embryo transfer, warm the blastocysts by submerging the vitrified droplet into a sucrose solution in base medium for , transfer and incubate in sucrose solution in base medium for , followed by two incubations in the base medium for .
?. [Cryopreservation by vitrification]
Leave the blastocysts to recover in standard Embryo-hold medium (with BSA), re-grade morphology according to IETS guidelines above and transfer within 2 hours.
?. [In vitro embryo culture]
On day five of culture, remove the embryos from the micro-wells and place them into single drops of LSOF and return to 7% O2 in modular incubation chamber. If aggregating embryos on day five, select all morulae (pre-compacting, compacting and compact) and go to step 9.
5 µl
Micro-well culture ends at this time point. As embryos develop into blastocysts, they expand to fill the micro-well and tightly adhere to its sides, so they can be easily damaged upon removal.
?. [In vitro embryo culture]
On day two of culture, refresh half of the medium () with warm, gassed ESOF to reduce the accumulation of ammonium and return to 7% O2 in modular incubation chamber.
10 µl
?. [Enucleation of zona-free oocytes]
Wash DNA-stained oocytes in H199+FBS and transfer into a H199+FBS droplet in the enucleation dish, covered by mineral oil, on the enucleation microscope stage ().
30 °C
?. [Collection of mature oocytes and zona removal]
Transfer oocytes into H199+BSA with caffeine. Maintain the oocytes in media containing caffeine before activation. Activate around 26.5 hpm, so include caffeine from 20.5 to 26.5 hpm.
The only steps that are carried out in media without caffeine are enucleation and fusion. In our experience, 20.5 hpm coincides with post-pronasing and recovery time.
?. [In vitro maturation of oocytes]
Dissect ovaries from the reproductive tract and place in a thermos in saline atfor aspiration within 2–4 hours of collection.
30 °C
?. [Collection of mature oocytes and zona removal]
Transfer zona-free oocytes in groups of 30 into drops in the 'oocyte plate' and hold them until enucleation.
?. [Chemical activation]
After 6-DMAP incubation, wash reconstructs thoroughly through two dishes of HSOF and culture as described below.
?. [Morula aggregation]
Pair morulae of similar developmental stage and morphological grade for aggregation.
?. [Collection of mature oocytes and zona removal]
Quickly wash oocytes through a fresh dish of H199+FBS to remove hyaluronidase. Then transfer to a dish of H199+BSA.
?. [Cell electrofusion (SCT)]
Wash fused reconstructs through HSOF-Ca2++10% FBS, and wash drops in 'ESOF-Ca plate', then transfer individual reconstructs to single drops (to prevent adhesion between reconstructs). Incubate at in a humidified 5% CO2 incubator with atmospheric O2 concentration.
5 µl
38.5 °C
?. [In vitro embryo culture]
Embryo culture ends on day six or seven before the blastocysts collapse and start degenerating. Embryo quality is morphologically graded using modified International Embryo Transfer Society (IETS) guidelines. Briefly, grade 1 embryos are excellent/good and have greater than 85% of their cellular material intact, and a large, regular inner cell mass (ICM); grade 2 are fair with some moderate irregularities but still have a robust ICM; grade 3 are poor, with major irregularities and/or little ICM.
?. [Chemical activation]
To activate, collect all of the reconstructs with a pipette and deposit into a dish containing freshly prepared solution of ionomycin in HSOF+1 mg/mL BSA for , followed by a wash through HSOF+30 mg/ml BSA for at least 3 minutes.
We recommended using the ionomycin solution within 10-30 minutes of thawing.
?. [Cryopreservation by vitrification]
Place the hook containing the droplet of embryos onto the surface of a stainless-steel cooling block (which has been equilibrated in a bath of liquid N2 for 15–20 seconds. Vitrification should be carried out within 60 seconds from the first exposure to the final vitrification solution, usually occurring after around 45 seconds.