Selection of donor nuclei in somatic cell-mediated gene transfer using a Co-transfection method

Hairong Ma, Zili Lu, Yi Sun, Tao Peng, Zhiqiang Shuai, Yufang Ma, Yan Zhang, Liang Wang, Xu Cao, Hanqing Wang

Research output: Contribution to journalArticle

Abstract

In this study, we introduced a co-transfection method for the selection of donor nuclei in somatic cell-mediated nuclear transfer. Two vectors were constructed in our experiment. One was pMSCV-GFP carrying the neomycin-resistant gene (Neor) and the green fluorescent protein (GFP) reporter gene; the other was pBC1-GFP carrying the mammary gland-specific promoter and target gene GFP. Ovine adult fibroblasts were co-transfected with pMSCV-GFP and pBC1-GFP. The data from this work demonstrated that the GFP genes in both vectors could successfully co-integrate into the genomes of ovine adult fibroblasts in three of the four transgenic cell clones assayed. Furthermore, PCR analysis of transgenic embryos proved that the GFP genes in both vectors could cointegrate into the genomes of the reconstructed embryos. Subsequently, analysis of the developmental rate of the reconstructed embryos after nuclear transfer indicated that the blastocyst rate from the co-transfected donor cells was similar (approximate 8 percent) to that from individual pMSCV-GFP transfected donor cells. The influence of co-transfection resulting in modification of donor nuclei on development of reconstructed embryos was also investigated. The results of flow cytometric analysis indicated that the co-transfected ovine fibroblasts had similar quiescent characteristics in terms of cell cycle (G0+G1 percent: 73.20 ± 4.04) to the individual pMSCV-GFP transfected fibroblasts (G0+G1 percent: 70.77 ± 1.19) after they were treated with serum starvation for five days. Our results suggest that the co-transfection method can be used for selection of donor cell clones in somatic cell-mediated gene transfer experiments. It can be potentially extended to applications related to expression of functional protein in mammary glands and other transgenic research relevant to nuclear transfer.

Original languageEnglish (US)
Pages (from-to)95-104
Number of pages10
JournalJournal of Reproduction and Development
Volume53
Issue number1
DOIs
StatePublished - Feb 1 2007
Externally publishedYes

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Keywords

  • Cell cycle
  • Co-transfection
  • Mammary gland-specific promoter
  • Ovine adult fibroblast
  • Somatic cell-mediated nuclear transfer

ASJC Scopus subject areas

  • Animal Science and Zoology

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