應用體細胞核移植技術在家畜育種及生物醫學之研究(1/3)
Date Issued
2005
Date
2005
Author(s)
DOI
932317B002021
Abstract
The purposes of this study in the first year
was to proceed with the cryopreservation of ear
skin fibroblast cells derived from elite pigs’
herds are able to march forward continuously
for cloning of elite pigs as well as to generate
of EGFP (enhanced green fluorescent protein)
transgenic pigs harboring EGFP marker gene
by pronuclear microinjection for isolation of
mescenchymal stem cells (MSC) of single
clone derived from their bone marrow. In our
results, primary fibroblast cells of
top-performance individuals were established
from skin samples taken from ear tissues. So
far, derived somatic cells from over 50 elite
pigs containing golden couples had been
cryopreserved in liquid nitrogen tank. In
addition, pig oocytes derived from ovaries of
sow, cumulus-oocyte complexes were matured
in vitro for 42–44 h, and oocytes with 1st polar
body were selected for enucleation and
reconstructed embryos could successfully
activated by electrical activation and the
electrical pulse combined with CB+ CHX or
6-DMAP would greatly improve the rates of
cleavage to 80-90% and blastocyst formation
rate to 30-40%. To study the effects of different
donor cell types and activation methods on the
subsequent development of porcine
reconstructed embryos produced by somatic
cell nuclear transfer (SCNT). A single
suspended fibroblast or mesenchymal stem cell
(MSC) was injected into enucleated ooplasm,
and reconstructed embroys were activated by
one of following methods: (1) electrical pulse
(EP, 10 sec at 5 V AC followed by a 1 x 30
µsec pulse at 2.2 kV/cm DC) combined with 10
µg/mL cytochalasin B and 10 µg/mL
cycloheximide for 4 h (EP+ CB+ CHX), or (2)
EP combined with 2 mM 6-DMAP for 4 h
(EP+ 6-DMAP). After activation treatments,
the reconstructed embryos were thoroughly
washed and cultured for 4 days in NCSU-23
medium. Results showed that the percentage of
blastocyst formation in these treatments using
the same activation method were greater in
Fibroblast groups than in MSC groups (37 vs.
34%, 28 vs. 24%). The rates of cleavage and
blastocyst formation were better in the groups
treated with EP+ 6-DMAP (88-89%, 34-37%)
than EP+ CB+ CHX (75-80%, 24-28%). Base
on upper result, we generated successfully 9
cloned piglets in one litter by nuclear transfer
of somatic cell derived from ear tissue of
top-performance boar from core of artificial
insemination. The efficiency of generating
cloned piglet is elevated to 4 % (9/223) from
0.5 % (4/795). This result indicated the
probability of this cloning technique for
commercialization. Additionally, enhanced
green fluorescent protein (EGFP) gene was
introduced into mouse genome of FVB and
ICR strain embryos by pronuclear
microinjection. A total of 326 FVB and 60 ICR
survival embryos were transferred into 12 and
2 recipient mice, respectively, and 68 FVB and
15 ICR pups were born. Nine (13 %) FVB and
four (27 %) ICR mice were confirmed to be
transgenic mice by PCR and Southern blotting
analysis. Among them, six FVB (9 %, 2♂/ 4♀)
and four ICR (27 %, 2♂/ 2♀) TG mice could
express EGFP fluorescent and exhibit stable
integration of the transgene in their germ line.
As the experiments of EGFP transgenic mice,
two transgenic fetuses of pig were produced
and the EGFP expression pattern is similar to
the transgenic mouse.
The EGFP expression pattern of transgenic
mice and pig fetuses was analysis by 365 nm
UV light. Exclusion of spleen, lung and bone
tissue, other organs (heart, muscle, brain, liver,
testis, gut, kidney, eyes and pancreas etc.)
highly expressed EGFP in transgenic mice and
pig fetuses. Bone marrow was flushed out from
femurs and tibias of transgenic candidates, the
near-to confluence fibroblastic colony were
obtained under the conditions of 2 X 106/cm2 of
the starting culture and the incubation time of
72 hours. Expression of EGFP in isolated
MSCs of transgenic pig fetuses and mice were
detected by using fluorescent microscope. The
EGFP MSCs derived from transgenic pig
fetuses and mice were preliminary isolated
from the transgenics in this study.
Progressively, the MSCs were successfully
conducted to give rise to adipocyte under
defined culture conditions. In conclusions, our
results indicates that the porcine reconstructed
embryos using MSC as donor cell exhibit the
poorer developmental potential in vitro, and the
activation method of EP+ 6-DAMP was
sufficient for cloned embryos to develop to
term in our studies. The cell populations will be
available in preclinical studies including cell
therapy, gene therapy, tissue engineering fields
and application in pig model.
Subjects
Somatic Cells
Nuclear Transfer
Cloned pigs
In Vitro Maturation
Porcine Oocytes
Mesenchymal
stem cells
stem cells
Publisher
臺北市：國立臺灣大學動物科學技術學系暨研究所
Type
report
File(s)![Thumbnail Image]()
Loading...
Name
932317B002021.pdf
Size
434.07 KB
Format
Adobe PDF
Checksum
(MD5):b7355beb07ca8304d5a64a386ed05ac1