Pronuclear visibility, development and transgene expression in IVM/IVF-derived porcine embryos

H. M. Kubisch, M. A. Larson, Hiroaki Funahashi, B. N. Day, R. M. Roberts

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

A total of 1550 zygotes was used to assess the timing of pronuclear visibility, embryo development following DNA microinjection, and transgene expression in IVM/IVF-generated porcine embryos. After centrifugation, pronuclei could be seen in 61.6% of zygotes. In 55.3% of these only 1 pronucleus was visible. Pronuclear visibility was highest at 20 h post-insemination. Zygotes were microinjected with 1 of 2 LacZ gene constructs driven by either the SV40 early promoter (pSVON) or the human cytoplasmic β actin promoter (pbActinLacZ). Development and transgene expression were assessed after either 48 h or 7 d in culture. After 48 h, significantly more zygotes with a single visible pronucleus developed to the 8-cell stage than zygotes in which no pronucleus had been seen (43.0 vs 24.8%), while those with 2 pronuclei were intermediate (31.4%). After 7 d, no difference in development to the morula stage was observed between noninjected control embryos (25.5%) and embryos with 1 (21.0%) or 2 pronuclei (22.5%); however, the proportion of embryos reaching the morula stage in the nonpronuclear group was significantly reduced (9.1%). After 48 h in culture, transgene expression was significantly higher in embryos with 2 pronuclei at the time of injection than in those with 1 (36.4 vs 17.9%). After 7 d in culture, 41.5% of morulae derived from zygotes with 2 pronuclei and 29.97% of thsoe derived from zygotes with 1 pronucleus showed signs of transgene expression. At this stage, significantly more morulae expressed the pbActinLacZ than the pSVON transgene (43.8 vs 25.8%). More than 80% of putative transgenic morulae or blastocysts showed evidence of mosaicism. These results demonstrate that IVM/IVF porcine embryos are able to develop in culture and express a microinjected transgene.

Original languageEnglish
Pages (from-to)391-401
Number of pages11
JournalTheriogenology
Volume44
Issue number3
DOIs
Publication statusPublished - 1995
Externally publishedYes

Fingerprint

pronucleus
Zygote
Transgenes
Morula
transgenes
zygote
embryo (animal)
Swine
Embryonic Structures
morula
swine
Lac Operon
Mosaicism
Insemination
promoter regions
Microinjections
Blastocyst
Centrifugation
Embryonic Development
Actins

Keywords

  • in vitro fertilization
  • in vitro maturation
  • LacZ
  • porcine embryos
  • transgenic expression

ASJC Scopus subject areas

  • Animal Science and Zoology
  • Equine
  • Food Animals
  • Small Animals

Cite this

Pronuclear visibility, development and transgene expression in IVM/IVF-derived porcine embryos. / Kubisch, H. M.; Larson, M. A.; Funahashi, Hiroaki; Day, B. N.; Roberts, R. M.

In: Theriogenology, Vol. 44, No. 3, 1995, p. 391-401.

Research output: Contribution to journalArticle

Kubisch, H. M. ; Larson, M. A. ; Funahashi, Hiroaki ; Day, B. N. ; Roberts, R. M. / Pronuclear visibility, development and transgene expression in IVM/IVF-derived porcine embryos. In: Theriogenology. 1995 ; Vol. 44, No. 3. pp. 391-401.
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AU - Roberts, R. M.

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AB - A total of 1550 zygotes was used to assess the timing of pronuclear visibility, embryo development following DNA microinjection, and transgene expression in IVM/IVF-generated porcine embryos. After centrifugation, pronuclei could be seen in 61.6% of zygotes. In 55.3% of these only 1 pronucleus was visible. Pronuclear visibility was highest at 20 h post-insemination. Zygotes were microinjected with 1 of 2 LacZ gene constructs driven by either the SV40 early promoter (pSVON) or the human cytoplasmic β actin promoter (pbActinLacZ). Development and transgene expression were assessed after either 48 h or 7 d in culture. After 48 h, significantly more zygotes with a single visible pronucleus developed to the 8-cell stage than zygotes in which no pronucleus had been seen (43.0 vs 24.8%), while those with 2 pronuclei were intermediate (31.4%). After 7 d, no difference in development to the morula stage was observed between noninjected control embryos (25.5%) and embryos with 1 (21.0%) or 2 pronuclei (22.5%); however, the proportion of embryos reaching the morula stage in the nonpronuclear group was significantly reduced (9.1%). After 48 h in culture, transgene expression was significantly higher in embryos with 2 pronuclei at the time of injection than in those with 1 (36.4 vs 17.9%). After 7 d in culture, 41.5% of morulae derived from zygotes with 2 pronuclei and 29.97% of thsoe derived from zygotes with 1 pronucleus showed signs of transgene expression. At this stage, significantly more morulae expressed the pbActinLacZ than the pSVON transgene (43.8 vs 25.8%). More than 80% of putative transgenic morulae or blastocysts showed evidence of mosaicism. These results demonstrate that IVM/IVF porcine embryos are able to develop in culture and express a microinjected transgene.

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