Comparison of electro-fusion and intracytoplasmic nuclear injection methods in pig cloning.
This paper methodologically compares the electro-fusion (EF) and intracytoplasmic injection (ICI) methods, as well as simultaneous fusion/activation (SA) and delayed activation (DA), in somatic nuclear transfer in pigs using fetal fibroblast cells. Comparison of the remodeling pattern of donor nuclei after nuclear transfer by ICI or EF showed that a high rate (80-100%) of premature chromosome condensation occurred in both cases whether or not Ca2+ was present in the fusion medium. Formation of pseudo-pronuclei tended to be lower for nuclear transfer performed by the ICI method (65% vs. 85-97%, p < 0.05). In vitro developmental potential of nuclear transfer embryos reconstructed with IVM oocytes using the EF method was higher than that of those produced by the ICI method (blastocyst formation: 19 vs. 5%, p < 0.05), and it was not improved using in vivo-matured oocytes as recipient cytoplasts. Embryos produced using SA protocol developed to blastocysts with the same degree of efficiency as those produced under the DA protocol (11 vs. 12%). Use of the EF method in conjunction with SA was shown to be an efficient method for producing cloned pigs based on producing a cloned normal pig fetus. However, subtle differences in nuclear remodeling patterns between the SA and DA protocols may imply variations in their nuclear reprogramming efficiency.
Kurome
Mayuko
M
Laboratory of Developmental Engineering, Department of Life Science, Meiji University, 1-1-1 Higashimita, Tama, Kawasaki 214-8571, Japan.
Fujimura
Tatsuya
T
Murakami
Hiroshi
H
Takahagi
Yoichi
Y
Wako
Naohiro
N
Ochiai
Takashi
T
Miyazaki
Koji
K
Nagashima
Hiroshi
H
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002459:Cell Fusion; D002875:Chromosomes / Q000235:genetics; D019976:Cloning, Organism / Q000379:methods*; D003593:Cytoplasm; D018274:Electroporation / Q000379:methods*; D004624:Embryo Transfer; D005260:Female; D007621:Karyotyping; D008297:Male; D008845:Microinjections / Q000379:methods*; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000502:physiology; D011247:Pregnancy; D013552:Swine
D002118:Calcium