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 language | English (US) |
|---|---|
| Pages (from-to) | 95-104 |
| Number of pages | 10 |
| Journal | Journal of Reproduction and Development |
| Volume | 53 |
| Issue number | 1 |
| DOIs | |
| State | Published - Feb 2007 |
| Externally published | Yes |
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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
- Reproductive Medicine
Cite this
Selection of donor nuclei in somatic cell-mediated gene transfer using a Co-transfection method. / Ma, Hairong; Lu, Zili; Sun, Yi; Peng, Tao; Shuai, Zhiqiang; Ma, Yufang; Zhang, Yan; Wang, Liang; Cao, Xu; Wang, Hanqing.
In: Journal of Reproduction and Development, Vol. 53, No. 1, 02.2007, p. 95-104.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Selection of donor nuclei in somatic cell-mediated gene transfer using a Co-transfection method
AU - Ma, Hairong
AU - Lu, Zili
AU - Sun, Yi
AU - Peng, Tao
AU - Shuai, Zhiqiang
AU - Ma, Yufang
AU - Zhang, Yan
AU - Wang, Liang
AU - Cao, Xu
AU - Wang, Hanqing
PY - 2007/2
Y1 - 2007/2
N2 - 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.
AB - 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.
KW - Cell cycle
KW - Co-transfection
KW - Mammary gland-specific promoter
KW - Ovine adult fibroblast
KW - Somatic cell-mediated nuclear transfer
UR - http://www.scopus.com/inward/record.url?scp=33947118617&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33947118617&partnerID=8YFLogxK
U2 - 10.1262/jrd.18082
DO - 10.1262/jrd.18082
M3 - Article
C2 - 17077582
AN - SCOPUS:33947118617
VL - 53
SP - 95
EP - 104
JO - Journal of Reproduction and Development
JF - Journal of Reproduction and Development
SN - 0916-8818
IS - 1
ER -