Abstract
Production of transgenic animals by somatic cell nuclear transfer (SCNT) has
a great value in the field of agriculture, biotechnology and human medicine. The
objective of the present study was to improve the in vitro development of bovine
transgenic cloned embryos. In Chapter I, the in vitro blastocyst formation rate was
significantly (P<0.05) higher (22.1%) in embryos derived from non-transfected
ear fibroblasts than that of human prourokinase (ProU) gene with green
fluorescent protein (GFP) marker gene transfected counterpart (10.3%). However,
the in vitro blastocyst formation rate (10.3 vs. 11.3%) did not vary in embryos
derived from either early (3-7) or late (8-12) passage transfected donor cells. In
Chapter II, the in vitro development of non-transgenic and transgenic cloned
embryos derived from human &1-antitrypsin (a1AT) gene with GFP marker gene
transfected cumulus cells did not vary significantly between modified synthetic
oviduct fluid (mSOF) and potassium simplex optimization medium (KSOM)
supplemented with BSA. The blastocyst formation rate in non-transgenic cloned
embryos was significantly (P<0.05) higher (33.3 to 38.5%) in BSA or FBS
supplemented KSOM than that in PVA supplemented counterpart (20.5%).
However, the in vitro developmental rate in transgenic cloned embryos did not
vary among BSA, FBS and PVA supplemented KSOM. In Chapter III, when
bovine fetal fibroblast (BFF) transfected with prion protein (PrP) mutant gene
with GFP marker gene (PrP-GFP) was fused with either metaphase II or telophase
II enucleated oocyte, the in vitro developmental rate in embryos did not vary
between the stages of recipient oocytes. When PrP-GFP or PrP-Ab (Prion protein
knock-out gene with antibiotic resistance marker gene) gene transfected BFF was
used as either frozen-thawed or cultured condition for reconstruction of embryos
with metaphase II enucleated oocytes, there were no significant differences in
blastocyst formation rates between two conditions of donor cells. However,
cloned embryos reconstructed with PrP-Ab transfected BFF developed at a higher
rate than that of PrP-GFP transfected counterparts (19.9 to 24.8% vs. 7.6 to
10.8%; P<0.05). Moreover, significantly (P<0.05) higher blastocysts formation
rates (34.8%) in transgenic cloned embryos reconstructed with PrP-Ab transfected
BFF were observed after supplementation of BSA in serum-free KSOM than that
after supplementation of PVA (20.0%). The GFP expression was observed in all
stages of embryo development from fused oocyte to blastocyst stage, and the
expression rate in blastocyst was 79% (27/34) with 11% (3/27) mosaic expression.
In Chapter IV, supplementation of 1.5 mM fructose in presence of 0.2 mM
glucose significantly improved the blastocyst formation rate in embryos
reconstructed with PrP-GFP transfected BFF (10.0 vs. 19.2%). This study
demonstrates that ear fibroblast transfection with ProU gene with GFP marker
negatively affects the in vitro development of bovine transgenic cloned embryos
although passage number (up to 12) of donor cells has no influence on that. Both
mSOF and KSOM are equally effective for in vitro culture of bovine transgenic
cloned embryos. However, response to protein supplementation in KSOM differs
between non-transgenic and a1 AT (with GFP marker) transgenic cloned embryos.
Both metaphase II and telophase II enucleated oocytes, and both frozen-thawed
and cultured donor cells can be used for reconstruction of bovine transgenic
cloned embryos. Selection of transfected donor cells by antibiotic is better than
GFP for in vitro development of cloned embryos. Supplementation of BSA in
serum-free KSOM and supplementation of fructose in combination with glucose
in chemically defined protein-free KSOM improve the in vitro development of
bovine PrP gene knock-out (with antibiotic resistance marker gene) and PrP gene
mutant (with GFP marker gene) cloned embryos, respectively.
Key Words: a1-antitrypsin, GFP, fructose, KSOM, prion protein, prourokinase,
transgenic cloned.
Student Number: 2001-31174