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 2007
Externally publishedYes

Fingerprint

Donor Selection
transfection
Green Fluorescent Proteins
green fluorescent protein
somatic cells
gene transfer
Transfection
Genes
fibroblasts
Fibroblasts
Sheep
methodology
embryo (animal)
Embryonic Structures
genetically modified organisms
Human Mammary Glands
sheep
mammary glands
genes
Clone Cells

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 journalArticle

Ma, Hairong ; Lu, Zili ; Sun, Yi ; Peng, Tao ; Shuai, Zhiqiang ; Ma, Yufang ; Zhang, Yan ; Wang, Liang ; Cao, Xu ; Wang, Hanqing. / Selection of donor nuclei in somatic cell-mediated gene transfer using a Co-transfection method. In: Journal of Reproduction and Development. 2007 ; Vol. 53, No. 1. pp. 95-104.
@article{0226c44b992b46b2a90b8c8f1adb61db,
title = "Selection of donor nuclei in somatic cell-mediated gene transfer using a Co-transfection method",
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.",
keywords = "Cell cycle, Co-transfection, Mammary gland-specific promoter, Ovine adult fibroblast, Somatic cell-mediated nuclear transfer",
author = "Hairong Ma and Zili Lu and Yi Sun and Tao Peng and Zhiqiang Shuai and Yufang Ma and Yan Zhang and Liang Wang and Xu Cao and Hanqing Wang",
year = "2007",
month = "2",
doi = "10.1262/jrd.18082",
language = "English (US)",
volume = "53",
pages = "95--104",
journal = "Journal of Reproduction and Development",
issn = "0916-8818",
publisher = "Japanese Society of Animal Reproduction (JSAR)",
number = "1",

}

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 -