Roslin Institute
Roslin
Midlothian EH25 9PS
UK
Telephone: 0131-527-4239
Fax: 0131-440-0434
E-mail: Keith.Campbell @bbsrc.ac.uk
Nuclear Transfer from an Established Cell Line
Keith H.S.Campbell, William A. Ritchie and Patricia A. Femer.
The technique of embryo reconstruction was first proposed
by Spemann (1938) to study the process of cell differentiation.
By transferring a single nucleus from cells of increasingly advanced
embryonic stages to an enucleated one cell embryo these experiments
were designed to determine at which point the developmental potential
of nuclei became restricted. If a somatic nucleus when transferred
into an enucleated egg can support development to term, then this
provides proof that there has been no loss or permanent inactivation
of genes leading to the formation of the somatic cell in question.
These experiments were first performed in the frog, Rana pipiens
(Briggs and King, 1952) and subsequently in the toad, Xenopus
laevis (Gurdon, 1974).Their findings led to the current concept
that equivalent, totipotent nuclei from a single individual could,
when transferred to an enucleated egg, give rise to genetically
identical individuals. In mammals similar procedures for "nuclear
transfer" were established by McGrath and Solter (1983).
Although it is now a decade since these procedures were first
applied in farm animals (Willadsen, 1986)the production of live
offspring has until recently, only been reported when embryonic
blastomeres or inner cell mass cells were used as nuclear donors
(for review see Campbell and Wilmut, 1997). We have recently described
the production of live offspring from a primary embryo derived
cell line (Campbell et al., 1 996b) .The use of cultured
cells as nuclear donors for embryo production provides the opportunity
to apply in farm animal species those techniques which have been
utilised in mice, via embryonic stem cells, for precise genetic
germ line modification (for review see Campbell and Wilmut, 1997).
The development of embryos reconstructed by nuclear transfer is
dependent on a large number of factors ( for reviews see Campbell
and Wilmut, 1994; Campbell et aL, 1996a) ). In the method
described here, the role of the donor cell type is unknown. The
important feature appears to be the induction of quiescence in
the donor cell. This may render the chromatin more amenable to
'interaction' with the recipient cytoplasm such that it is able
to control development in the spatial and temporal manner required
(for discussion see Campbell ,Wilmut, 1997). For this reason we
have not included the method for the isolation of the c~l type
previously reported(Campbell et aL, 1 996b).
For embryo manipulation a Nikon Diaphot TDM inverted microscope
equipped with Differential Interference Contrast and epi-Fluorescence
is used. This is fitted with 2 x Narishige MO-188 Hydraulic Joystick
Micro manipulators and 2 x Narishige IM-188 micro injectors which
are modified to accept gas tight syringes (Figurel). A 500 ~il
Hamilton syringe (Sigma S 0142) is mounted on the right hand side
of the microscope in order to control the holding pipette mounted
on the left hand side of the stage. A similar syringe, but with
a capacity of 250 al~ is mounted on the left hand side to control
the enucleation pipette mounted on the right hand side of the
stage. The injection system is modified by the addition of a two
way stopcock (Vigon VG1) to the syringe barrels which is then
connected with tubing to the micro-tool holders, this allows a
5 mi syringe to be used as a Hydraulic reservoir (Figure 2). The
system is filled with Fluorinert FC 77 (Sigma F- 4758).
For the preparation of manipulation pipettes, glass capillaries
(Cat. No CC 10-100 and Cat.
No GC1OO-T15) are obtalned from Clark Electromedical Instruments.
A Campden Instruments
Moving Coil Micro electrode puller (Model 753) is used in conjunction
with a Micro forge
(Research Instruments MF 1). For grinding of pipettes a rotating
wheel which fits into the micro
forge was home made (4cm diameter, rotation speed of 20-60 r.p.m.).
Aluminium oxide paper in sizes 0.3, 1.0, and 3.Omm was obtained
from Microfinishing Systems Project Industrial Abrasives Division/3M(
223-6N 3M Center. St Paul, MN 55144, USA). In addition an extra
tool holder attached by a length of silicone tubing to a SOmi
syringe for producing an air flow whilst grinding pipettes is
required.
For cell fusion a home made pulse generator was constructed, this
is capable of producing an AC pulse up to 5V for 20 seconds followed
by up to 4 DC pulses of 10-200 ~sec duration, ranging from 0.25
to 2.0 kV/cm when using a fusion chamber with electrodes 200 ~un
apart. The fusion chamber, again home made, consists of two electrodes
of 100 ~nn diameter platinum wire glued to the bottom of a 4.5
cm glass petri dish with a gap of 200 um
For all embryo handling Wild M8 and M3Z dissecting microscopes
fitted with home made heated stages are used. In addition the
following pieces of equipment are required; an electronic thermometer
(PTM1) Petracourt Ltd; Drummond model 105 (5~il) and 110 (l0~il)
pipettes; an automatic pipette Gilson 200~; glass chips measuring
4mm x 25mm cut from 2mm thick glass.
For embryo manipulation and culture Fluorinert FC 77 (Cat.No.
F4758), NaCl ( Cat.N0
S5886), KCl (Cat. N0. P5405), CaCl2..2H20 (Cat. N0. C7902), KH2PO4(Cat.
N0. P5655),
MgSO4.7H20 (Cat. N0. M1880), NaHCO3 (Cat. N0. S5761), HEPES (Cat.N0.
H9136), Sodium
lactate (Cat. N0. L7900), Sodium pyruvate (Cat. N0. P5280), Glucose
(Cat. N0. G6138), BSA
(Cat.N0. A6003), Penicillin G Potassium Salt (Cat. N0. P4687),
Streptomycin sulphate (Cat. N0.
S1277), Phenol Red (Cat.N0. P0290), Cytochalasin B (Cat.N0. C6762),
Sigmacote' ( S12),
Hydrofluoric acid, Tween 80 (Cat.N0. P1754), Hyaluronidase W-S
(Cat.N0. H3884), bis-Benzimide (Hoechst 33342) (Cat.N0. B2261),
Manitol (Cat.N0. ), TC 199 (Cat.N0. M0148), Dimethyl sulfoxide
(DMSO) (Cat. N0. D4540) and M2 medium (Cat N0 M1767) were obtained
from SIGMA Ltd. Dow Coming Silicone Fluid 200/50 cs (Cat.N0. 63006
4V), hard paraffin wax (Cat N0~ 29839) Decon 90 (Cat. N0. 56022)
were obtained from BDH. Petroleum Jelly 'Vaseline' from Cheesebrough
Ponds Ltd, PBS tablets (Dulbecco 'A') (Cat N0. BR14a) from Oxoid,
Foetal Calf Serum (Cat N0. 29-101- 49) from ICN Biomedicals Ltd.
Agar (Cat N0 0140-01) was purchased from Difco Ltd. NB. Hydrofluoric
acid antidote gel (Cat N0 H1430190) was obtained from Fischer
Scientific UK.For the synchroniation and hyperstimulation of ~wes
to be used as oocyte donors, temporary and final recipients oFSH
'Ovagen' was purchased from Immuno-chemicals Ltd New Zealand,
Sponges 'Veramix' from Upjohn Ltd, PMSG 'Folligon' from Intervet
and GnRH 'Receptal' from Hoechst UK Ltd. Sutures 'Dexon' were
obtained from Davis and Geck Ltd. For culture and assessment of
donor cells BH}C21 (Glasgows MEM. Cat No.21710-025), 200 mM L-Glutamine
(Cat N0. 25030-024), lOomNiSodium Pyruvate (Cat N0 11360-039),
Minimal Essential Medium (MEM Cat N0. 11140-035), Colcemid l0~g/mi
(Cat N0. 15212-012), Trypsin 2.5% (Cat N0. 25090-028) were
obtained from GIBCO Life Technologies. Sodium chloride (Cat N0.
102414J), di Sodium Phosphate (Cat N0. 103834G), Potassium di
Hydrogen Orthophosphate (Cat N0. 102032W), Potassium Chloride
(Cat N0. 101938K), D-Glucose (Cat N0. 101 174Y), TRIS (Cat N0.
103 154M), Giemsa R66 Solution (Cat N0. 350864X) were purchased
from BDH LTD. EGTA (Cat N0. E4378), Polyvinyl Alcohol (Cat N0.
P8136) and Phenol Red (Cat N0. P-0290) were purchased from SIGMA
LTD, Methanol (Cat N0. M/4000/17), Acetone (Cat N0. A'0600/17)
and Glacial Acetic Acid (Cat N0. A/0400/PB 17) from Fisons LTD
and Foetal Calf Serum from Globepharm. Human anti-PCNA seurm and
RITC conjugated rabbit anti-human antiserum from DAKOPATTS. The
mountant used was Vecta shield (Cat NO. HlOOO) from Vector Labs.
Two types of pipettes are used, one for holding the oocyte
and the other for manipulation.
1. Holding pipette: (Figure 3)
Steps
1. Pull glass capillaries (GC1O-100) by hand over a very small
flame to give a diameter of l00-l50 um
2. Mount a drawn capillary onto the micro forge. Apply heat and
make the first bend close to the start of the pulled part of the
pipette at an angle of 450
3. Make a second bend approximately 1 cm from the first making
it parallel to the rest of the capillary.
4.2 cm from the second bend a third bend is made at 450 and
a fourth close to the last bend to make the glass parallel to
the rest of the pipette again.
5. Cut the pipette 2 mm from the final bend by using a diamond
pencil to mark the glass. Make sure that the break is at right
angles to the pipette.
6. Place the broken end of the pipette over the filament on the
micro forge. Apply heat until the open tip is almost closed ensuring
a smooth end with a diameter of approximately 20~im.
2. Enucleation Pipette: (Figure 4)
Steps
1. Pull glass capillaries (GC1OO T15) using the Moving Coil
Micro electrode puller to give an initial taper which reduces
the diameter of the capillary to slightly greater than the diameter
required, the second taper being almost parallel.
2. Mount a drawn capillary on the micro forge. Measure the diameter
of the pipette using an eyepiece graticule and break at the required
size, usually between 15-25piii. The break is made by fusing the
capillary onto a bead of glass which is heated using the minimum
temperature required to allow the glass to stick onto the capillary.
The power is then turned off and the filament pulled down rapidly
breaking the pipette. Care should be taken to ensure that the
capillary is not overheated as this may cause distortion and thickening
of the glass. The tip area must be as straight as possible so
that it can be easily ground to a point.
3. Mount the pipette in an instrument holder attached to the micro
forge and pass a continuous flow of air through the it, using
a SOmi syringe and length of tubing. This airflow prevents dirt
from entering the pipette as it is ground. A turntable mounted
on the micro forge allows an aluminium oxide disc with a grit
size of 0.3, 1.0 or 3.0um to grind the pipette. The grit size
depending on the diameter of the pipette required. The pipette
should be ground at an angle of 450 Care should be taken
to ensure that the pipette is fully ground as problems can arise
if grinding is incomplete and an unground edge is left. A pipette
which is not completely ground is impossible to sharpen.
4. Mount the ground pipette in a micro-tool holder attached to
a length of tubing.
5. Clean the external surface by dipping into 20% Hydrofluoric
acid for 30 to 60 seconds whilst continuously blowing air through
(this prevents acid from entering the inside of the pipette).
6. Wash with distilled water to remove the acid.
7. Mount the pipette in the micro forge horizontally, with the
hole in view. Heat the forge to the minimum temperature required
to melt the glass bead. Touch the tip against the glass bead and
pull the tip of the pipette out to a sharp point.
II). MANIPULATION CHAMBER: (Figure 5)
Materials and Solutions
1. Petroleum jelly: wax mixture: mix 9g petroleum jelly
and 1 g hard paraffin wax in a SOmi glass beaker. Heat gently
until melted and thoroughly mixed then decant into 10 mi syringes
and allow to cool.
2. Cytochalasin B stock solution: Add DMSO to cytochalasin
to make a lmg/mi solution.
3. M2 without calcium and magnesium: Dissolve the following
components in 800mi of milli-Q water KCl 0.356g, CaCl2.2H20 0.252g,
KH2PO4 0. 162g, MgSO4.7H20 0.293g, NaHCO3 0.349g, HEPES 4.969g,
Sodium Lactate 2.610g or 4.349 mi of 60% syrup, Sodium pyruvate
0.036g, Glucose 1 .OOOg, BSA 4.OOOg, Penicillin G 0.060g, Streptomycin
sulphate 0.050g, Phenol red 0.OlOg. Adjust ph to 7.4 and osmolality
to 28OmOsm make up to 1 litre with milli-Q water.
4. Siliconised glass slides: Good quality glass slides
are washed thoroughly in Decon, rinsed five times in distilled
H20 and dried over night in a hot air oven. The washed and dried
slides are then dipped in 'Sigmacote', drained of excess fluid
and oven dried.
Steps
1 Apply a line of petroleum jelly/wax mixture approximately
2.5cm in length along both edges of the upper surface of a siliconised
glass slide.
2. Attach a glass chip (4mm x 25mm cut from 2mm thick glass) to
each line of the mixture and press well down.
3. Apply petroleum jelly/wax mixture to the top of each glass
chip.
4. Clean a glass coverslip with 70% ethanol.
5. Pipette 300 mi of manipulation medium into the centre of the
chamber. Place the cleaned coverslip onto the mixture and push
down to make a water-tight seal.
6. Pipette 200 al of Dow Corning Silicone Fluid 200/50 cs into
each end of the chamber. This seals the ends, preventing changes
in osmolality and also allows the entry of the micro tools.
B. PREPARARTION OF EQUIPMENT FOR MANIPULATION.
Solutions
1.1.25% Tween 80: l.25mi Tween 80 in 98.75mi of 2X distilled
water.
Steps
1. Place a prepared manipulation chamber onto the stage of
the microscope.
2. Attach the holding pipette to the left hand side tool holder
ensuring that all air is removed from the hydraulic system.
3. Move the holding pipette into the manipulation chamber and
draw a small volume of manipulation medium into it.
4. Wash the inside of the enucleation pipette thoroughly in a
solution of 1.25 % Tween 80, by aspirating the solution through
the pipette several times.
5. Coat the inside of the pipette with foetal calf serum, using
the same method. This will prevent the cytoplasm from sticking
to the pipette.
6. Mount the pipette on the right side of the chamber, and ensure
that the system is free from air bubbles. A slight angle, of approximately
50 from horizontal, allows the pipette to pick up cells from the
bottom of the chamber.
C. ENUCLEATION OF OOCYTES
Solutions and equipment.
1. Manipulation chamber and micro-tools: (as described
in section Al & All)
2. Mu oocytes: (as described in section G).
3. Hyaluronidase stock solution: Dissolve hyaluronidase
in PBS to give 15,000 IU/mi. Dispense into 20~il (300 IU) aliquots.
Store at -200C.
4. M2 medium without calcium and magnesium: (see section
II)
5. Cytochalsin B Stock solution: (see section II).
6. Bis-Benzimide (Hoescht 33342) stock solution :1 .Omg/ml
in distilled water, prepare l0~il aliquots store at -200C.
7. M2 medium: (SIGMA)
Steps
1. Remove the cumulus cells from the oocytes by placing them
into Calcium free M2 without FCS containing 600 units/mi of Hyaluronidase.
Incubate for 10 minutes at 370C and then repeatedly pipette with
an automatic pipette (Gilson 200~il) until the cumulus cells are
removed.
2. Wash oocytes in calcium free M2/l0% FCS until all of the cumulus
cells are removed from the medium.
3. At regular intervals place batches of 10 to 15 oocytes into
Calcium free M2 10% FCS containing 7.5~g/mi Cytochalasin B plus
5 ~g/mi bis-Benzimide and culture at 370C for 15 minutes.
4. After incubation transfer each batch of treated oocytes to
the manipulation chamber.
5. With 40x magnification pick up and attach a single oocyte to
the holding pipette using negative pressure.
6. Move the manipulation chamber so that the remainder of the
oocytes are no longer in the field of view.
7. Change the magnification to 200x DIC and focus on the oocyte
held by the pipette.
8. Bring the enucleation pipette into focus. Using the enucleation
pipette rotate the oocyte into a position where the polar body
plus the area of cytoplasm adjacent to it can be aspirated into
the pipette (see Figure 6a).
9. Increase the suction on the holding pipette to hold the oocyte
more securely, then insert the enucleation pipette through the
zona pelucida at a p6int opposite the holding pipette.
10. Manipulate the enucleation pipette into a position next to
the polar body, apply a small amount of negative pressure and
aspirate the polar body and a small amount of cytoplasm from directiY
beneath it into the pipette (see Figure 6b).
11. Withdraw the pipette from the oocyte, then remove the oocyte
from the field of view.
12. Turn off the transmitted light source, change to UV illumination
(blue light) and examine the aspirated karyoplast (whilst inside
the pipette) for fluorescence using filter block UV2A. If the
metaphase has been removed it will fluoresce with a blue colour,
the polar body will also be visible. The metaphase plate fluoresces
with a lower intensity than the polar body (see Figure 6c).
13. Move the enucleated oocyte to the right hand side of the chamber
and discard the aspirated karyoplast from the pipette. If the
enucleation was unsuccessful then it is possible to repeat the
procedure a second time.
14. Remove completed batches of oocytes from the manipulation
chamber and place into a micro drop or dish containing calcium
free M2 10%FCS. Maintain at 370C until all the oocytes have been
enucleated.
D. EMBRYO RECONSTRUCTION.
Solutions and equipment.
4.Fusion Medium: 0.3 M manitol with the addition of
0.1 mM MgSO4 and 0.05 mM CaCl2 in 2x distilled H20 with an osmolority
of 280 mOsm.
5. Fusion Chamber: Consisting of two 100~un diameter platinum
wires glued to the bottom of a 4.5 cm glass petri dish at a distance
of 200~im apart.
Steps
1. Prepare a manipulation chamber, as for the enucleation
procedure, containing calcium free M2 10%FCS.
2. Using a hand drawn capillary mouth pipette, place the nuclear
donor cells into the upper right hand corner of the chamber and
a group of enucleated oocytes into the centre of the chamber.
3. Pick up an oocyte with the holding pipette.
4. Move the chamber to the top right side.
5. Focus the microscope onto the bottom of the chamber.
6. Move the enucleation pipette to the bottom of the chamber,
manoeuvre it to a suitable cell, and genfly aspirate it into the
pipette (see Figure 6d).
7. Re-focus onto the enucleated oocyte and move the enucleation
pipette until it is in focus.
8. Insert the enucleation pipette through the hole previously
made in the zona pelucida. Whilst holding the pipette against
the cytoplasm expel the donor cell into the perivitelline space(see
Figure 6e). Ensure contact between the two cells of the couplet
(see Figure 61). 9. Donor cells are transferred to groups of 10
oocytes. Upon completion of each batch electrofusion is carried
out as soon as possible.
10. Wash the cytoplast/karyoplast couplets in SOOmi of warm (370C)
fusion medium.
11. Place 80~ of fusion medium, spanning the electrodes, into
the fusion chamber.
12. Pipette the batch of cytoplast/karyoplast couplets into the
fusion medium but outside the electrodes and allow to settle.
13. Place a couplet between the electrodes, using a hand drawn
capillary mouth pipette, moving the couplet until the plane of
contact between cytoplast and karyoplast is parallel to the electrodes
(see Figure 7). Precise orientation is necessary for fusion to
occur.
14. Apply the fusion pulse which consists of a S second AC pulse
of 0.15kV/cm followed by three DC pulses of 1.25 kV/cm for 80
~ec.
15. Remove the couplets from the chamber and place each batch
into a 20~il drop of TC 199
10% FCS containing 7.5 ~g/mi Cytochalasin B. Incubate at 370C
for 1 hr.
16. Wash the couplets in TC 199 10% FCS without cytochalasin,
transfer to M2 and examine for cell fusion.
17. Transfer fused couplets to 20~ drops of TC199 10%FCS under
oil, culture overnight at
370C.
E. CULTURE OF RECONSTRUCTED EMBRYOS IN LIGATED OVIDUCTS
OF TEMPORARY RECIPIENT EWES
Solutions
1. Molten agar 1.2 % w/v: Add 1.2 g agar in loomi of
PBS (Dulbeccos A), heat until dissolved.
2. Recipient Ewe: (see section F).
Steps
1. Place molten agar into embryo culture dishes and cool to
370C. The temperature is monitored using an electronic thermometer.
The agar is maintained at 370C using a heated stage fitted to
the dissecting microscope.
2. When the agar has cooled to 370C, place one or two of the fused
embryos into the agar. Suck a small amount of agar containing
the embryos into a Drummond model 105, 5~d pipette.
3. Remove the pipette from the agar and allow to cool.
4. Place the tip of the pipette into cool M2 (at room temperature)
and expel the agar cylinder formed into the medium.
5.Trim the agar cylinders containing the embryos using 2 x 25
gauge needles attached to 2 x imi syringe barrels.
F. SYNCHRONISATION OF EWES FOR OOCYTE RECOVERY AND AS TEMPORARY AND FINAL RECIPIENTS.
Solutions
1. oFSH 1 unit in 16 mi (2ml/ewe/injection, subcutaneously)
2.Veramix sponges
3. GnRH Receptal (2ml/ewe intramuscularly)
4. PMSG Folligon 500 units (0.5 mi/ewe intramuscularly)
4. Suture Dexon 5 metric.
Steps
1. Superovulation and synchronisation regimes.
Oocyte donors
Sponge in 0
oFSH 8am 10
oFSH 5pm 10
oFSH 8am 11
oFSH 5pm 11
oFSH 8am 12
oFSH 5pm 12
oFSH Sponge out 8am 13
oFSH 5pm 13
GnRH 7am 14
Heat detection and starve
Oocyte recovery am 15
Temporary recipients
Sponge in 0
Sponge out + PMSG 12
Heat detection 14
Starve 15
Embryo transfer 16
Starve 21
Embryo recovery 22
Final recipients
Sponge in 0
Sponge out 8am 13
Heat detection 15
Starve 21
Surgery 22
G. RECOVERY OF OVULATED OOCYTES FROM OOCYTE DONORS.
Solutions and Equipment.
1. Catheter: a short piece of plastic tubing of suitable
diameter used to enter the oviduct of the ewe allows the oocytes
to be flushed from the animal. Sterilise by immersing in ethanol.
2. PBS/]%FCS: PBS (Dulbecco A) (Oxoid BRl4a) tablets made
up with 2x distilled water. Add lOmi FCS /1.
Steps
1. Anaesthetise a super ovulated donor ewe using Sodium Intraval
and perform a mid line lap arotomy.
2. Exteriorise the uterus to expose the ovaries and oviducts.
3. Place a small catheter into the fimbria. Using a blunt 18 gauge
needle introduced close to the uterotubal junction, flush 20 mi
of warm (370C) PBS/FCS through each oviduct. Collect the flushings
in an embryo collection dish.
4. Examine the flushings under the dissecting microscope, transfer
cumulus oocyte complexes to fresh calcium free M2 and keep at
370C in an atmosphere of 5% C02 in air.
H. TRANSFER TO AND RECOVERY FROM TEMPORARY RECIPIENT EWES OF AGAR CHIPPED EMBRYOS
Solutions
Steps
1. Anaesthetise a pseudo-pregnant ewe using Sodium Intraval,
intubate and carry out a mid ventral laperotomy.
2. Place the agar embedded embryos into M2 medium at 370C.
3.Using Dexon suture, double ligate each oviduct near the uterotubal
junction.
4. Pick up the agar cylinders containing the embryos in a small
volume of medium using a lOal Drummond pipette.
5. Carefully insert the tip of the pipette into the fimbria end
of the ligated oviduct.
6. Push the end of the pipette as far down the oviduct as possible
using a pair of fine nontoothed forceps to grasp the oviduct.
7 Carefully expel the agar cylinders into the oviduct. Suture
the incision and allow the ewe to recover.
8. At day 7 (from the time of embryo reconstruction) euthanase
the recipient and recover the oviducts.
9. Dissect excess tissue from the oviducts and remove the ligatures.
10. Flush each oviduct towards the utero-tubule junction with
Smi of M2 medium using a syringe and a blunted 18 gauge needle.
11. Collect the flushings in an embryo dish. Examine under the
dissecting microscope and locate the agar cylinders.
12. Wash the agar cylinders in M2 and assess embryo development.
13. Dissect morulaiblastocyst stage embryos from the agar using
2 x 25 gauge hypodermic needles attached to 1 mi syringe barrels.
H. TRANSFER OF EMBRYOS TO FINAL RECIPIENT EWES.
Steps
1. Anaesthetise a day 7 pseudo-pregnant ewe and perform a
mid line laparotomy.
2. Pick up 2 or 3 of the recovered morula/ blastocyst stage embryos
in a small volume of M2 using long form pasteur pipette with a
flame polished end.
3. Make a small hole in the wall of the uterus using a blunt 18
gauge needle.
4. Insert the end of the pipette carrying the embryos into this
hole and expel the embryos into the uterus.
5. Suture incision and allow the ewe to recover.
6. Ultrasound scan the recipient ewes after 45 days to determine
pregnancy.
General Solutions
1.Culture Medium: To SOOmis of BHK-21 (Glasgows MEM) add 56mis
of FCS, 5.6 mis of L-Glutamine, 5.6mis of Sodium Pyruvate and
5.6mis of MEM-non essential amino acids.
2.Trypsin Solution: To prepare a working solution dissolve
6.3g Sodium Chloride, 0. 12g di Sodium Phosphate, 0.21 6g potassium
dihydrogen Orthophosphate, 0.333g Potassium Chloride, 0.9g D-Glucose,
2.7g TRIS and 0.8mis of Phenol Red in 800mi of milli-Q water.
Add to this lOOmis of Trypsin solution (2.5%), 0.4g EGTA and 0.lg
PVA. Adjust the pH to 7.6 and make up to lOOQmi. Filter sterilise,
aliquot and store at -200C.
K; CULTURE OF CELLS FOR MITOTIC ARREST
Solutions
Colcemid: Add 2~il of stock solution per mi of medium.
Steps
1. Take an exponentially growing culture of donor cells which
has reached approximately
80% confluence (75cm2 culture flask).
2. Aspirate growth medium and wash 2x with warm (370C) PBS (Dulbecco's
A).
3. Remove excess liquid, add 2mi of trypsin solution, ensure even
covering of cell monolayer.
4. Incubate at 370C, monitor the cell culture for detachment of
cells.
5. When cells are detached add 10 mi of warm culture medium.
6. Decant cells into a universal container and pellet at 1000
rpm for 5 minutes.
7 Remove medium and resuspend cell pellet in lOmi of fresh warm
medium.
8.Add 2mi of cell suspension and 8 mi of growth medium to a
75cm flask.
9.Incubate for 48 hours.
10. After 48 hours change the growth medium and incubate for 2
hours.
11. Add 40~~ of colcemid stock solution per 19.96 mis of growth
medium and continue
incubation overnight.
12. Harvest cells and prepare spreads as below.
L. PREPARATION OF MITOTIC SPREADS.
Solutions:
1 Trypsin: Working stock (see Section J)
2. Hypotonic KCl (0.56% w/v): Dissolve 5.6g KCl in lOomi
milli-Q water.
3. Methanol:Acetic acid fixative: Prepare freshly, mix
18 mi of methanol with 6 mi of Glacial Acetic Acid, cool and keep
at 40C for use.
Steps
serum.
minutes.
6.Remove as much of the liquid as possible.
10.Mix contents of the tube by inverting once then leave at
room temperature for 5 minutes.
15.Airdry.
M. INDUCTION AND ASSESSMENT OF QUIESCENCE IN DONOR CELLS
Solutions
1. Culture medium: (see Section J)
2. Low serum culture medium: Prepare lx medium as previous
(Section J) but containing
0.5% FCS (replace volume with milli-Q water).
3.Clean sterile coverslips: Wash 22mm diameter coversups
by boiling in 1 litre of 1.0% Decon for 10 minutes. Wash lOx in
tap water and lOx in milli-Q water. Remove excess liquid by blotting
onto clean absorbent towelling then air dry. Sterilise in a hot
air oven at 1800C for 4 hrs.
Steps
1. Trypsinise an 80% confluent monolayer of donor cells and
resuspend in growth medium.
2.Replate cells at 1/5th the original density in growth medium
and culture for 48 hours.
3. Wash monolayer Sx with low serum medium then continue culture
in this medium.
4. Exit from the growth cycle and survival in the quiescent state
must be established for each cell line. In the same culture add
a number of cleaned coversups. At the time of serum removal and
at 24 hour intervals remove a coverslip and process for PCNA staining
as detailed below.
N. STAINING FOR PCNA
Solutions
1 .Methanol:Ace tone Fixative: Prepare freshly a 50:50
mixture of methanol:acetone. Cool to
4 C and store on ice for use.
2. Humidified Chamber: Place a sheet of filter paper into
a 9cm petri dish. Dampen with distilled water.
4.PBS/1% FCS: Add 100~il FCS to 10 mi sterile PBS.
5.1 Antibody working solution: Dilute PCNA antibody 1:10
with PBS/i %FCS.
6.20 Antibody working solution: Prepare 1:40 dilution of
FITC conjugated anti human in PBS/i %FCS. Microfuge l3OOxg for
30secs before use.
Steps
1. Remove coverslip from cell culture.
2. Rinse in PBS at 40C, then fix in Methanol:Acetone for 4 mins
at 40C.
3. Wash in PBS and keep in PBS until staining.
4. Remove excess liquid and transfer coverslip to a humidified
chamber.
5. Add 40~ 10 antibody solution to each coversup, incubate
at 40C overnight.
6. Wash 3x in PBS.
7. Remove excess liquid and add 40~ of 20 antibody. Incubate
at room temperature for 4 hrs.
8. Wash 3x in PBS.
9. Remove excess liquid place coverslip face down on a 5~ drop
of mountant, blot away excess and seal edges of coverslip with
nail vamish.
10. Keep at 40C in darkness.
11. Examine using epifluorescence for PCNA staining.
12. Record the percentage of PCNA +ve nuclei. PCNA is a nuclear
antigen visible during S- phase, the staining pattern is variable
for examples refer to Kill et al., (1991).
IV. Comments
The method described here is based on the reconstruction of
ovine embryos using a cultured cell line as nuclear donors. The
development of reconstructed embryos is dependent upon a large
number of factors however, in thls method the crucial factors
are the timing of the individual steps which may vary dependent
upon the breed of sheep used. In our experience using Scottish
Blackface ewes in Scotland oocyte recovery should be carried out
between 28 and 31 hours post GnRH injection. Enucleation as soon
as possible after recovery and embryo reconstruction 36-39 hours
after GnRH. The time required for the cells to be used as nuclear
donors to exit the growth
cycle and enter a quiescent or GO state is dependent upon individual
cell lines. This should be determined for each cell type to be
used.
V. Pitfalls
activated spontaneously as a result of handling will resume
meiosis, this will in general be visible
when the aspirated karyoplast is examined with the appearance
of a third area of fluorescence,
corresponding to the anaphase/telophase.
throughout the oocyte handling and manipulation procedures.
will hold. This prevents the embryos being lost into the uterus.
chip, some of the embryos will collapse. This makes assessment
difficult however, after culture for
1-2 hours in M2/l0%FCS embryos generally re-expand and assessment
is made easier.
cells.
Figure 1. Nikon microscope fitted with Narashige manipulators
for embryo manipulation.
Figure 2. Syringe reservoir and 3 way stop cock fitted
to modified syringes for the control of enucleation and holding
pipettes
Figure 4. Prepared manipulation chamber.
Figure 6. Oocyte enucleation and preparation of couplet prior to fusion. a) attchment of the oocyte to the holding pipette (note position of polar body. b) aspiration of karyoplast. c) identification of polar body (RHS) and metaphase plate (LHS) by epifluorescence. d) picking up a donor cell. e) and f) placing donor cell inside zona pellucida and ensuring contact of oocyte and donor cell.
References
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Kill, I.R., Bridger, J.M., Campbell, K.H.S., Maldonado-Codina, G. and Hutchison, C.J. (1991)The timing of the formation and usage of replicase clusters in S-phase nuclei of human diploid fibroblasts. J. Cell. Sci. ,100,869-876.
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Acknowledgements.
This work was supported by MAFF, BBSRC and and by grant n0
B O2CT-920358 from the
Biotechnology program of the EEC.