Developmental potential and transgene expression of porcine nuclear transfer embryos using somatic cells.
We examined whether porcine nuclear transfer (NT) embryos carrying somatic cells have a developmental potential and NT embryos carrying transformed fibroblasts express transgenes in the preimplantation stages. In Experiment 1, different activation methods were applied to NT embryos and the development rates were examined. Relative to A23187 only or A23187/6-DMAP, electrical pulse made a significant increase in both cleavage rate (58.1+/-13.9 or 60.7+/-6.3 vs. 74.9+/-7.5%) and development rate of NT embryos to the blastocyst stage (2.2+/-2.8 or 2.2+/-1.5 vs. 11.0+/-4.1%). In Experiment 2, in vitro developmental competence of NT embryos was investigated. The developmental rate to the blastocyst stage of NT embryos (9.9+/- 2.4% for cumulus cells and 9.8+/-1.6% for fibroblast cells) was significantly lower than that (22.9+/-3.5%) of IVF-derived embryos (P<0.01). NT blastocysts derived from either cumulus (28.9+/-11.4, n = 26) or fibroblast cells (30.2+/-9.9, n = 27) showed smaller mean nuclei numbers than IVF-derived blastocysts (38.6+/-10.4, n = 62) (P<0.05). In Experiment 3, nuclear transfer of porcine fibroblasts expressing the GFP (green fluorescent protein) gene resulted in green blastocysts without losing developmental potential. These results suggest that porcine embryos reconstructed by somatic cell nuclear transfer are capable of developing to preimplantation stage. We conclude that somatic cells expressing exogenous genes can be used as nuclei donors in the production of NT-mediated transgenic pig.
Koo
D B
DB
Korea Research Institute of Bioscience and Biotechnology, Taejon, Korea.
Kang
Y K
YK
Choi
Y H
YH
Park
J S
JS
Kim
H N
HN
Kim
T
T
Lee
K K
KK
Han
Y M
YM
D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D004558:Electric Stimulation; D047108:Embryonic Development; D005314:Embryonic and Fetal Development; D005260:Female; D005307:Fertilization in Vitro; D005347:Fibroblasts / Q000166:cytology; D015870:Gene Expression; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics; D008297:Male; D053652:Nuclear Transfer Techniques; D011247:Pregnancy; D013552:Swine; D019076:Transgenes
D007476:Ionophores; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D000001:Calcimycin; C001020:N(6),N(6)-dimethyladenine; D000225:Adenine