Production of viable cloned miniature pigs by aggregation of handmade cloned embryos at the 4-cell stage

Reprod Fertil Dev. 2014 Mar;26(3):395-406. doi: 10.1071/RD12243.

Abstract

The aim of the present study was to improve the quality of handmade cloned porcine embryos by multiple embryo aggregations. Embryos derived from aggregation of three cloned embryos (3×) had a better blastocyst rate than cloned control (1×) embryos (73.6% vs 35.1%, respectively; P<0.05), but did not differ from those produced by aggregation of two cloned embryos (2×; 63.0%). Total cell numbers differed among treatments (P<0.05), with the greatest cell numbers (126) in the 3× group and the lowest (55) in the control group. The ratio of inner cell mass:total cell number was comparable in the 2× and 3× groups (25.1% vs 26.1%, respectively) and was significantly better than that in the control group (15.3%). The proportion of apoptotic cells in 2× and 3× groups was lower than that in the control group (2.7% and 2.2% vs 4.7%, respectively; P<0.05). Expression of Oct4 and Cdx2 was higher, whereas that of Bax was lower (P<0.05), in the 3× compared with non-aggregate group. Seven piglets were born to two surrogate mothers after embryo transfer of 3× aggregated blastocysts. In conclusion, aggregated embryos had greater total cell numbers and better pluripotency gene expression, with reduced expression of the pro-apoptosis gene Bax. Collectively, these improvement may be associated with the development of cloned embryos to term.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Analysis of Variance
  • Animals
  • Apoptosis / physiology
  • Blastocyst Inner Cell Mass / cytology
  • CDX2 Transcription Factor
  • Cell Aggregation / physiology*
  • Cloning, Organism / methods
  • Cloning, Organism / veterinary*
  • DNA Primers / genetics
  • Embryo, Mammalian / embryology*
  • Female
  • Gene Expression Profiling
  • Homeodomain Proteins / metabolism
  • Octamer Transcription Factor-3 / metabolism
  • Pregnancy
  • Pregnancy Outcome
  • Real-Time Polymerase Chain Reaction
  • Swine
  • Swine, Miniature / embryology*
  • Trans-Activators / metabolism
  • bcl-2-Associated X Protein / metabolism

Substances

  • CDX2 Transcription Factor
  • DNA Primers
  • Homeodomain Proteins
  • Octamer Transcription Factor-3
  • Trans-Activators
  • bcl-2-Associated X Protein