Production of porcine cloned transgenic embryos expressing green fluorescent protein by somatic cell nuclear transfer.
In the present study, nuclear transferred embryos (NTEs) were reconstructed by using pig fetal fibroblasts as donors and in vitro matured oocytes as recipients. The effects of G418 selection on donor cells, duration of IVM of prepubertal gilt oocytes and oxygen tension in IVM of oocytes were investigated. The results were as follows: (i) When G418 selected cells expressing GFP were used as donors, the cleavage rate of NTEs decreased drastically in comparison to NTEs derived from donors without antibiotic selection (47.5% vs. 71.6%, p < 0.05). For the blastocyst rate, no significant difference was observed between two groups (10% vs. 10.4%, p > 0.05). (ii) The rate of nuclear maturation of oocytes increased significantly when IVM duration time was extended from 36 to 42 h (83.6% vs. 96.7%, p < 0.05). However, no statistical difference was observed between NTEs derived from oocytes of 36 h IVM group and NTEs from oocytes of 42 h IVM group in the rates of cleavage (59.3% vs. 73.6%, p > 0.05) and blastocyst formation (9.3% vs. 13.2%, p > 0.05); (iii) no significant difference was observed between NTEs reconstructed from oocytes matured under lower oxygen (7% O2) tension and NTEs derived from oocytes matured under higher oxygen tension (20% O2) in cleavage rate (70.6% vs. 67.1%, p > 0.05) and blastocyst rate (11.8% vs. 12.3%, p > 0.05). These results suggest that: (i) G418 selection does not have a significant effect on cleavage rate of NTEs expressing GFP. (ii) Nuclear maturation is greatly improved by prolonging IVM duration from 36 to 42 h, while no significant differences were observed for developmental potential of transgenic embryos. Thus IVM 42 h is the better choice in order to obtain maximum number of M II oocytes as recipients. (iii) Lower oxygen tension and higher oxygen tension in IVM have no significant effect on development of cloned embryos.
Zhang
Yunhai
Y
State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, China.
Pan
Dengke
D
Sun
Xiuzhu
X
Sun
Guojie
G
Wang
Xiaobo
X
Liu
Xiaohui
X
Li
Yan
Y
Dai
Yunping
Y
Li
Ning
N
D000818:Animals; D030801:Animals, Genetically Modified; D001757:Blastomeres / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D019976:Cloning, Organism / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D046149:Embryo Culture Techniques / Q000379:methods; D004624:Embryo Transfer; D004622:Embryo, Mammalian / Q000378:metabolism*; D005260:Female; D005307:Fertilization in Vitro; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D010100:Oxygen / Q000494:pharmacology; D013552:Swine / Q000196:embryology / Q000235:genetics*; D013997:Time Factors; D014162:Transfection
D049452:Green Fluorescent Proteins; D010100:Oxygen