Production of cloned pigs from cultured fetal fibroblast cells.
Somatic cell nuclear transfer was used to produce live piglets from cultured fetal fibroblast cells. This was achieved by exposing donor cell nuclei to oocyte cytoplasm for approximately 3 h before activation by chemical means. Initially, an experiment was performed to optimize a cell fusion system that prevented concurrent activation in the majority of recipient cytoplasts. Cultured fibroblast cells were fused in medium with or without calcium into enucleated oocytes flushed from superovulated gilts. Cybrids fused in the presence of calcium cleaved at a significantly (P < 0.05) greater rate (69%, 37 out of 54) after 2 days of culture compared with those fused without calcium (10%, 7 out of 73), suggesting that calcium-free conditions are needed to avoid activation in the majority of recipient cytoplasts during fusion. In the second experiment, cybrids fused in calcium-free medium were activated approximately 3 h later with ionomycin, followed by incubation in 6-dimethylaminopurine to determine development in vitro. Following 2 days of culture, cleavage rates of chemically activated and unactivated cybrids (fusion without activation control) were 93% (100 out of 108) and 7% (2 out of 27), respectively. After an additional 5 days of culture, activated cloned embryos formed blastocysts at a rate of 23% (25 out of 108) with an average inner cell mass and trophectoderm cell number of 10 (range, 3 to 38) and 31 (range, 16 to 58), respectively. In the third experiment, activated nuclear transfer embryos were transferred to the uteri of synchronized recipients after 3 days of culture to assess their development in vivo. Of 10 recipients receiving an average of 80 cleaved embryos (range, 40 to 107), 5 became pregnant (50%) as determined by ultrasound between Day 25 and Day 35 of gestation. Of the five pregnant recipients, two subsequently farrowed one piglet per litter originating from two different cell culture lines. In this study, efficient reprogramming of porcine donor nuclei by fusing cells in the absence of calcium followed by chemical activation of recipient cytoplasts was reflected in high rates of development to blastocyst and pregnancy initiation leading to full term development.
Boquest
Andrew C
AC
BresaGen Limited, P.O. Box 259, Rundle Mall, Adelaide, South Australia 5000, Australia. andrew.boquest@adelaide.edu.au
Grupen
Christopher G
CG
Harrison
Sharon J
SJ
McIlfatrick
Stephen M
SM
Ashman
Rodney J
RJ
d'Apice
Anthony J F
AJ
Nottle
Mark B
MB
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002459:Cell Fusion; D002467:Cell Nucleus / Q000502:physiology; D002478:Cells, Cultured; D019976:Cloning, Organism; D004396:Coloring Agents; D005260:Female; D005333:Fetus / Q000166:cytology / Q000502:physiology*; D005347:Fibroblasts; D006824:Hybridization, Genetic; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008297:Male; D018895:Microsatellite Repeats; D009865:Oocytes / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine
D004396:Coloring Agents; D007476:Ionophores; D015759:Ionomycin; D002118:Calcium