Annual Report 2006

Fig. 5
PGC and EBC fused cells
PGCs and EBCs were were fused using inactivated HVJ (A-I) or electrical stimulation (J and K). Cells were stained
with Hoechst 33342 to visualise their nuclei (B, D, F, I), and incubated for 2 hours (E, J, K) or 16 hours (A, C, H).
Photographs E and F are merged and shown in photograph G. PGC and EBC fused cells were identified by their
morphology and difference in size of the two nuclei. Scale bar represents 10 µm.
mean fusion rate of PGC with EBC using
inactivated HVJ was 0.96%. When cell fusion
was carried out using electrical stimulation,
pearl chains were formed within 20 seconds by
exposing cells to the AC field. After applying
DC pulses, adjacent cells in a pearl chain were
fused in some places. Fused cells of more than
three cells were also occasionally observed. The
fused cells of PGC and EBC were shown in Fig.
5 (J and K). The mean fusion rate of PGC with
EBC using electrical stimulation was 5.2%. The
system for producing viable offspring derived
from nuclear transferred PGCs makes it
possible to manipulate the germline of chickens
through somatic cells.
Lack of αGal epitope in
transgenic cloned pigs by
expression of EndoGalC
Because organs of pigs are biologically and
anatomically similar to human organs, porcine
organs are expected to use as replacement for
human organs, which are chronically in short
supply. The major problem with
xenotransplantation of porcine organs into
humans is the hyperacute rejection caused by
the reactions involving natural human antibodies
and the complement system. It is considered
that natural antibodies against αGal epitope on
pig cells are the main cause of hyperacute
rejection. Recently, the overseas companies
succeeded to produce α1,3GT knocked out pigs
by somatic cell cloning for elimination of αGal
epitope.
The endo-β-galactosidase C (EndoGalC)
secreted from cleaved
αGal epitope from pig cells under physiological
pH conditions. In addition, transfection with
EndoGalC gene effectively reduced αGal
expression in cultured porcine aortic
endothelial cells. Thus, the cleavage of αGal
epitope by EndoGalC would be the alternative
to knock out α1,3GT gene for elimination of
αGal epitope in whole pigs.
The EndoGalC expression vector was
transfected in Meishan fetal fibroblasts by
electroporation. After selection under G418 for
10-14 days, the cells lacking αGal epitope were
separated and collected by FACS (fluorescentactivated
cell sorting). The separated cells were
cultured and expanded sufficient for nuclear
transfer to produce live piglets. 1144 of nuclear
transferred embryos at 2-8 cell stage were
transferred to 7 synchronized recipient pigs.
Two recipients were pregnant but one