l cloning in porcine:
in vitro and in vivo development of nuclear transfer complexes
The a cloning synchr as sta the nu
and, in some cases, the fusion rate.
of pro electric
standa 7.5 hours
ithout degradation. This was necessary in order to provide enough time to carry out
permit
a high embryos reaching the blastocyst stage.
the second set of experiments, a nuclear transfer protocol was developed which
stage efficien
numbe st morphology.
the third phase of this work, the viability of the embryos produced by the nuclear ansfer protocol was confirmed by transfer of activated zygotes to synchronized gilts.
his resulted in the birth of two viable piglets.
Michael Hölker
Experimental studies of somatic cel
derived from in vitro matured oocytes
im of the present study was to establish a protocol for porcine somatic cell and to produce viable piglets from reconstructed embryos transferred to onized gilts. In vitro matured oocytes out of slaughterhouse ovaries were used rting material for all experiments. The criteria for evaluating each protocol were mber of nuclei found at specific developmental stages, blastocyst morphology,
In the first set of experiments, a parthenogenetic model was used for rapid evaluation tocols for maintaining matured oocytes in culture and activating them by
al stimulation and DMAP. The result of this work was the establishment of a rd protocol which permitted the storage of embryos for periods up to
w
subsequent steps of the nuclear transfer procedure. This set of experiments also ted the optimization of the activation procedure to get a high cleavage rate and percentage of parthenogenetic
In
gave a satisfactory percentage of reconstructed embryos reaching the blastocyst during in vitro culture. This was accomplished by optimization of fusion cy (fusion rate) and by varying the duration of maturation while monitoring the r of nuclei at each developmental stage and blastocy
In tr T
The following results were obtained:
Using a standard two step maturation protocol, 71% of all oocytes maintained in the mat
1.
uration medium reached the metaphase II stage after 44 hours in culture, as evaluated by Hoechst staining.
2.
ytes cleaved and 29.6% of the activated oocytes reached the blastocyst stage. Other field strengths (1.25
s. 13.8%) and blastocyst rates (29.6% vs. 12.9% vs.
4.3% vs. 0%). The difference between the 0.8 KV/cm group and the 1.0 KV/cm
3. ing parthenogenetic embryos, the standard one step NCSU23 culture system gave significantly higher cleavage rates than a three step culture
. Storage of oocytes for 7.5 hours before activation in TL-Hepes resulted in a
5.
antly higher blastocyst rates after activation than storage in TL-Hepes with an In experiments with parthenogenetic embryos, the optimal electric field strength for a fixed pulse duration of 45 µsec was 1.0 KV/cm. With this combination, 72.9% of all activated ooc
KV/cm, 1.5 KV/cm, and 1.75 KV/cm) gave significantly lower cleavage (72.9%
vs. 60.5% vs. 25.9% v
group was not significant.
Again us
system consisting of: sequential use of 1) cr2 for 48 hours; 2) NCSU23+BSA for 48 hours; and 3) NCSU23+FCS for 72 hours (62.9% vs. 54.7%).
4
significantly higher number of nuclei at blastocyst stage than storage in Hb-NCSU23 (22.3% vs. 18.7%).
In a further experiment with parthenogenetic embryos, storage over 7.5h in TL-Hepes with an osmolarity of 296 mOsmol or 321 mOsmol gave signific
osmolarity of 271 mOsmol (18.3% vs. 24.4-26.2%).
6. In a comparison between electrostimulation alone and the combination of electroactivation followed by a second stimulation with 6-DMAP; the two step combination gave a significantly higher rate of blastocyst formation (40.4% vs.
26.5%).
. In a fusion experiment, two calcium free media, Ca free SOR2 medium and Boquest fusion medium, resulted in lower precocious activation (cleavage)
oblast-oocyte complexes matured for 38-40 hours (83.8% vs. 75.5%).
1. In nuclear transfer experiments, maturation of oocytes for 40 hours resulted in a highly significant improvement in development to the blastocyst stage as compared to oocyte maturation for either 38 hours or 42 hours (9.6% vs.
14.8% vs. 4.9%). This was not seen in the parthenogenetic control groups in which there was no exposure-time effect observed in the blastocyst rate.
12. After transfer of cloned embryos produced from in vitro matured oocytes, two of four recipient gilts established a pregnancy. One gilt farrowed and gave birth to four piglets of which two remain in riting.
7
rates after administration of the electrical pulse than the calcium containing Sor 2 medium (8.9% vs. 6.7% vs. 29.1%).
8. Spontaneous activation (cleavage) of the porcine oocytes without an electric field was only 1.0%.
9. Fusion of fibroblast-oocyte complexes in Ca free Sor 2 medium resulted in significantly higher fusion rates than in Boquest fusion medium (84.2% vs.
67.1%).
10. Fusion of fibroblast-oocyte complexes matured for 42-43 hours gave significantly higher fusion rates than fusion of fibr
1
good health at the time of w
13. The recipient gilt was excluded from being the biological mother by analysis of 12 highly polymorphic microsatellite loci selected from the set developed at the analysis confirmed that, among the four ed
In con he ults that it is possible to maintain the
develo pote al in TL-Hepes media with an
osmola 6 o 21 urs. This allows sufficient time to
perform en eps It is clear that additional
activation with DMAP can improve activation rates of reconstructed complexes.
Additio s on ion steps within the nuclear
transfe ithin a relatively short time window. The
develo s maintained in culture and the
birth o e functionality of the
nuclea ther changes in the composition of
media cond ns pient
oocyte ainl pro
University of Bonn. This microsatellite
piglets born, two were derived from one fibroblast line and two were deriv from a second fibroblast line.
clusion, t res of this work indicate pmental nti of porcine oocytes stored
rity of 29 r 3 mOsmol for up to 7.5 ho
subsequ t st in the nuclear transfer protocol.
nal result dem strate that micromanipulat r protocol have to be performed w
pment rates of nuclear transfer derived embryo
f live piglets after transfer to foster mothers demonstrate th r transfer protocol described here. Fur
and the itio during embryo culture to gain a better quality of reci s will cert y im ve the efficiency of this protocol.
8 A ng er
BSA Bovines Serum Albumin cAMP cyclic (zyklisches) Adenosin
CB Cytochalasin B
CBS Castrated Boar Serum CdK Cyclin dependent Kinase CSF Cytosolic Sperm Factor
CsK Cyclin stabilisierender Komplex
CYCLO Cyclohexim
DC Direct Chain (Gleichstrom)
d.h. das heißt
DMAP 6-Di-Methyl-Amino-DMEM Dulbecco’s Modified E DMSO Di-Methyl-Sulf-Oxid
DNA Desoxiribo
eCG equine Chorionic Gonadrotropin EDTA Ethylen-Diamin-Tetra-Acid
GTP Guanosine Tri Phoshat GV-Stadium Germinal-Vesikel-Stadiu
HAR Hyperakute Abstoßungs Reaktion hCG human Chorionic Gonadotropin
Hepes N-[2-Hydroxyethyl]piperazin-N’-[Ethansulfonsäure]
osphat
IE Internationale Einheiten Ins3P Inositol 1,4,5-Tri Ph IONO Ca-Ionophore
IVC In Vitro Culture (In-vitro-Kultur) IVF In Vitro Fertilizatio
IVM In Vitro Maturation (In-v IVP In Vitro Production
KDa KiloDalton
NCSU North Carolina State NEBD Nuclear Envelop B
PS Newborn Piglet S
p Probabilität (Irrtumswahrscheinlichkeit) PBS Phosphat Buffered Saline (Medium)
Pb Polar body (Polkörper)
omosome Condensation
Prostaglandin F2α
l 4,5-bis-Phosphat
rimordiale Keimzelle
MSG Pregnant Mare’s Serum Gonadotropin
Ribo Nucleotide Acid (Ribonukleinsäure)
Sekunde
tandardabweichung
erosal
PCC Premature Chr
PCR Polymerase Chain Reaction PGF2α
pFF porzine Follikel-Flüssigkeit PIP2 PhosphatidylInosito
TCM Tissue Culture Medium
TE TrophEktoderm
TIM Thim
Tl Tyrode’s Albumin Laktat Pyruvat (Medium)
TR Teilungs Rate
U Unit
V Volt
VNTR Variable Number of Tandem Repeats
vs. versus
µs Mikrosekunde
µm Mikrometer
Mittelwert x
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