Method of oocyte activation affects cloning efficiency in pigs.
The following experiments compared the efficiency of three fusion/activation protocols following somatic cell nuclear transfer (SCNT) with porcine somatic cells transfected with enhanced green fluorescent protein driven by the chicken beta-actin/rabbit beta-globin hybrid promoter (pCAGG-EGFP). The three protocols included electrical fusion/activation (NT1), electrical fusion/activation followed by treatment with a reversible proteasomal inhibitor MG132 (NT2) and electrical fusion in low Ca(2+) followed by chemical activation with thimerosal/dithiothreitol (NT3). Data were collected at Days 6, 12, 14, 30, and 114 of gestation. Fusion rates, blastocyst-stage mean cell numbers, recovery rates, and pregnancy rates were calculated and compared between protocols. Fusion rates were significantly higher for NT1 and NT2 compared to NT3 (P < 0.05). There was no significant difference in mean nuclear number. Pregnancy rate for NT2 was 100% (n = 19) at all stages collected and was significantly higher than NT1 (71.4%, n = 28; P < 0.05), but was not significantly higher than NT3 (82.6%, n = 23; P < 0.15). Recovery rates were calculated based on the number of embryos, conceptuses, fetuses, or piglets present at the time of collection, divided by the number of embryos transferred to the recipient gilts. Recovery rates between the three groups were not significantly different at any of the stages collected (P > 0.05). All fusion/activation treatments produced live, pCAGG-EGFP positive piglets from SCNT. Treatment with MG132 after fusion/activation of reconstructed porcine embryos was the most effective method when comparing the overall pregnancy rates. The beneficial effect of NT2 protocol may be due to the stimulation of proteasomes that infiltrate donor cell nucleus shortly after nuclear transfer.
Whitworth
Kristin M
KM
Division of Animal Sciences, University of Missouri, Columbia, Missouri 65211, USA.
Li
Rongfeng
R
Spate
Lee D
LD
Wax
David M
DM
Rieke
August
A
Whyte
Jeffrey J
JJ
Manandhar
Gaurishankar
G
Sutovsky
Miriam
M
Green
Jonathan A
JA
Sutovsky
Peter
P
Prather
Randall S
RS
D000704:Analysis of Variance; D000818:Animals; D001755:Blastocyst; D002118:Calcium / Q000494:pharmacology; D002467:Cell Nucleus; D065150:Cellular Reprogramming; D019976:Cloning, Organism / Q000379:methods*; D004558:Electric Stimulation; D004624:Embryo Transfer; D005260:Female; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D011247:Pregnancy; D018873:Pregnancy Rate; D013552:Swine / Q000235:genetics / Q000502:physiology*; D013849:Thimerosal / Q000494:pharmacology; D015053:Zygote / Q000378:metabolism
D007976:Leupeptins; D049452:Green Fluorescent Proteins; D013849:Thimerosal; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D002118:Calcium