11.20-13.20 Sperm nuclear
injection
Shoko Ishibashi, Lab 4.12, King Henry
Building
Sperm Nuclear Transplantation
Shoko Ishibashi1, Kristin L. Kroll2 and Enrique Amaya1
1
The Healing Foundation Centre, Faculty of Life Sciences, University of Manchester,
Manchester, United Kingdom and 2Department of Molecular Biology and Pharmacology,
Washington University School of Medicine, St Louis, MO, USA
INTRODUCTION
The transgenesis protocol is based on restriction enzyme mediated integration
(REMI), and can be divided into three parts, 1) preparation of high-speed egg extracts,
2) sperm nuclei preparation and 3) nuclear transplantation. This protocol describes a
method for the nuclear transplantation in Xenopus laevis. After brief incubation of
sperm nuclei with linearized plasmid DNA, egg extract and a small amount of the
restriction emzyme are added. The egg extracts partially decondenses chromosomes
and the restriction enzyme stimulates recombination by creating double-strand breaks,
facilitating integration of DNA into the genome. Diluted nuclei are transplanted into
unfertilized egg. Since the transgene integrates into the genome prior to fertilization,
the resulting transgenic embryos are not chimeric and there is no need to breed to the
next generation in order to obtain non-mosaic transgenic animals.
MATERIALS
Reagents
[R] Cysteine solution (2.5% in 1X MMR, pH 7.8-8.0)
Gentamycin (1000X; 10 mg/ml)
High-speed egg extract
Human Chorionic Gonadotropin (HCG, 1000 U/ml; store at 4˚C)
[R] Linearized plasmid (100 ng/µl)
Magnesium chloride (100 mM)
[R] MMR (Marc’s Modified Ringer; 0.1X)
[R] MMR (0.4X and 0.1X with 6% Ficoll/10 µg/ml Gentamycin)
Pregnant Mare Serum Gonadotropin (PMSG, 100 U/ml PMSG; store at –20˚C)
Restriction enzyme
[R] Sperm Dilution Buffer (SDB)
Sperm nuclei
Equipment
Injection dishes; 1.0% agarose in 0.1X MMR is poured into 60-mm Petri dishes.
Before the agarose solidifies, a template is laid onto it. After the agarose has
solidified, the templates are removed and dishes are wrapped in parafilm and stored at
4˚C until use. As a template we use a 35-mm X 35-mm weighing boat which holds
about 400 X. laevis eggs.
Transplantation Apparatus: Harvard Apparatus 22 syringe pump (NP 55-2222), two
2.5 ml Hamilton Gas Tight Syringes, plastic tubing (ID=0.7 mm, OD=2.4 mm) filled
with Mineral Oil (SIGMA, M8410) and micromanipulator (Fig.1A). Set the flow rate
at 0.6 µl/min.
Transplantation Needles: 30-µl Drummond MICROCAPS® (1-000-0300) are pulled
to produce large needles with long, gently sloping tips. We use a Micropipet Puller
Model P-87 (Sutter Instruments Co.) for pulling needles using a condition, p=50,
v=100 and t=5. Needles are clipped with a forceps to produce a beveled tip of 80-100
µm diameter, using the ocular micrometer of a dissecting microscope for
measurement (Fig.1B).
METHOD
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Prime two females 3-5 days before HCG injection with 50 units of PMSG.
Inject the females with 500 units of HCG, 12-15 hours before they are needed.
Allow an aliquot of SDB to equilibrate to room temperature, and make up 2.5%
Cysteine in 1X MMR, pH 8.0.
Turn on the switch of the Harvard Apparatus and start the infusion pump to get
the flow to stabilize before injection.
Set up a reaction using a clipped yellow tip: mix 4µl sperm stock (~4-8 X 105
nuclei) and 1-2µl linearized plasmid (100 ng/µl), and incubate for 5 min at room
temperature.
Dilute 0.5 µl of a restriction enzyme in 4.5 µl of water, and mix 1 µl of the
diluted enzyme with 18 µl of SDB, 2 µl of 100 mM MgCl2 and 2 µl of highspeed egg extract.
Add the mixture to the sperm/DNA and well by gentle pipetting (using a clipped
yellow tip). Incubate for 15 min at room temperature.
During the reaction, collect eggs in a beaker by squeezing frogs and dejelly them
in 2.5% Cysteine/1X MMR, pH 7.8-8.0. This usually takes about 10 min, so by
the time the eggs are ready, the reaction is nearly complete. Squeeze eggs directly
into a dry beaker and add Cysteine solution immediately to keep egg quality.
Wash the dejellied eggs with 1X MMR at least three times, and transfer the eggs
to injection dishes containing 0.4% MMR/6% Ficoll/10 µg/ml Gentamycin using
a wide-bore Pasteur pipet. We generally fill the square space with eggs so that no
gap is left between the eggs. Transplantation should be performed at around
16˚C. To achieve this we place the injection dish on the plastic box half-filled
with ice and place the box and eggs under the injection microscope.
After the incubation with extracts, mix the sperm nuclei gently by pipetting with
a clipped yellow tip. Then transfer 5 µl of the reaction into 150 µl of SDB that
has equilibrated at room temperature.
Mix well but avoid making bubbles, using a clipped yellow tip with a piece of
plastic tube attached (Fig. 2A). Fill the clipped yellow tip with the diluted sperm
suspension, carefully detach the clipped yellow tip, keeping the tip horizontal and
backfill a transplantation needle by attaching it to the tube (Fig. 2B). You can
keep the yellow tip with the remaining nuclei, by placing it horizontally, in case
you need to load another needle. Keep decondensed sperm nuclei at room
temperature and transplant them within an hour, but preferably within 30 min.
Attach the needle to the tube filled with mineral oil that is connected to the
syringe on the Harvard Apparatus.
Check the flow and start injecting. Keep the needle inside each egg for
approximately 0.5 second, and move the needle fairy rapidly from egg to egg
piercing the plasma membrane of each egg with single, sharp motion. We usually
transplant for about 15-20 min. If needle is blocked by debris during
transplantation, change needle or try to fix by pinching the tube or cutting the tip
of needle using forceps.
After injection, incubate embryos in injection dishes at 16˚C.
15. When the embryos reach the 4-cell stage (about 3-4 hours after injection at 16˚C),
gently transfer normally dividing embryos to 10-cm Petri dish containing 0.1X
MMR/6% Ficoll/10 µg/ml Gentamycin using a wide-bore Pasteur pipet.
16. The next day, when embryos are around stage 12, transfer healthy embryos to a
new 10-cm Petri dish containing 0.1X MMR/10 µg/ml Gentamycin. Because of
the large needle tip used for transplantations, embryos often develop large blebs
at the site of injection. These blebs occur when cells are forced out of the hole left
in the vitelline membrane at the injection site, but they generally do not affect
development falling off at the neurula or tailbud stages.
17. Incubate embryos until they reach the stage that you need to analyze at 14-22˚C.
TROUBLRSHOOTING
Problem: No cleaving eggs
[Step 15]
Solution: (1) make sure that injection needle is not blocked. (2) The dilution of the
sperm nuclei and the injection volume delivered during the transplantations are not
appropriate.
Problem: Many embryos die during gastrulation
[Step 16]
Solution: Try not to damage sperm nuclei during its preparation and reaction
Problem: Number of embryos expressing transgene is low
[Step 17]
Solution: (1) Make sure that the enzymes used for linearization and reaction do not
digest within your construct. (2) Increase the amount of enzyme for the reaction by
changing a dilution rate.
DISCUSSION
One person can transplant sperm nuclei into several hundred to thousands of eggs in a
typical experiment. About 30-40% of these transplanted eggs develop into normally
cleaving 4-cell stage embryos. About 60-80% of these embryos proceed through
gastrulation normally, while the other 20-40% exhibit gastrulation abnormalities
resulting from chromosomal damage to the sperm nuclei or physical damage to the
egg occurring during transplantation. Thus approximately 20-30% of the eggs initially
injected with nuclei proceed to post-gastrula stages, and approximately 10-50% of
these embryos show stable expression of transgenes. Since Xenopus embryos can be
obtained rapidly, at low cost, and in large numbers, this technique would be useful.
Transgenesis can be used in many applications; 1) to misexpress genes during
development, 2) to label specific structures, using the fluorescent protein as a marker
and 3) to study the regulation of genes. In this method, the transgene integrates into
the genome as a concatemer (5-35 copies; Kroll and Amaya 1996). Therefore two
different constructs mixed in the same reaction co-integrate into the same site of the
genome at high frequency (80-90%; Hartley et al. 2001). To distinguish transgenic
embryos, a marker gene, for example -crystallin promoter driving GFP, can be cointegrated with a desired transgene. In case where the effect of misexpression is
subtle, it may be difficult to rely solely on F0 transgenic embryos for the analysis. The
reason for this is that each F0 animal is unique; i.e. each carries a different copy
number of transgenes and distinct sites of integration.
REFERENCES
Hartley KO., Hardcastle Z., Friday RV., Amaya E., Papalopulu N. 2001. Transgenic
Xenopus embryos reveal that anterior neural development requires continued
suppression of BMP signaling after gastrulation. Dev Biol. 238: 168-184.
Kroll KL., Amaya E. 1996. Transgenic Xenopus embryos from sperm nuclear
transplantations reveal FGF signaling requirements during gastrulation. Development
122: 3173-3183.
Figure Legends
Figure 1. (A) We use an oil-filled injection system for the nuclear transplantations.
The syringe and tubing are filled with mineral oil and the infusion pump depresses the
syringe plunger, resulting in a constant, desirable flowrate. (B) Transplantation needle
has a gently sloping tip and is clipped with forceps to produce a beveled, 80-100 µm
wide tip.
Fig. 2. (A) The diluted reaction mix is drawn into a clipped yellow tip containing
about 0.5-1cm of Tygon tubing. (B) The yellow tip containing the Tygon tubing and
dilute sperm nuclei is carefully detached from the pipetman and connected to the back
of needle using the tubing. The needle is gently loaded with the dilute sperm nuclear
reaction by gravity. This is done by slowly increasing the angle of the yellow
tip/tubing/needle so that the mixture flows gently into the needle. Once the needle is
complete filled, the needle is detached from the yellow tip and the needle is ready to
connect to the infusion pump. The remaining sperm mixture can be set aside
horizontally and used to reload another needle, if two people are injecting
simultaneously or if a needle is accidentally damaged or blocked.
Recipe
Cysteine
2.5%(w/v) L-Cysteine hydrochloride 1-hydrate in 1X MMR (Sperm Nuclear
Transplantation)
Adjust pH with NaOH to 7.8-8.0. Prepare before use.
Dithiothreitol (DTT), 100 mM
filter-sterilize and store in aliquots at –20˚C
Linearized plasmid
Any enzyme can be used for linearization of plasmid. Digest DNA using standard
conditions, and purify by phenol/chloroform extraction and ethanol precipitation.
There is no need to gel purify the plasmid.
MMR, 10X
1 M NaCl2
20 mM KCl
10 mM MgCl2
20 mM CaCl2
50 mM HEPES (pH 7.5)
Adjust pH with NaOH to 7.5. Sterilize solutions by autoclaving.
SDB
250 mM sucrose
75 mM KCl
0.5 mM spermidine trihydrochloride
0.2 mM spermine tetrahydrochloride
Add about 80 µl of 0.1N NaOH per 20 ml solution to titrate to pH 7.3-7.5 and store
0.5-1 ml aliquots at –20˚C.
Spermidine trihydrochloride, 10 mM
filter-sterilize and store aliquots at –20˚C
Spermine tetrahydrochloride, 10 mM
filter-sterilize and store aliquots at –20˚C
Sucrose, 1.5M
filter-sterilize and store in aliquots at –20˚C