Efficiency of gene transfection into donor cells for nuclear transfer of bovine embryos

The production of transgenic (TG) animals by somatic cell nuclear transfer (SCNT) has proven to be a more efficient method than other methods, such as gene injection or sperm mediation. The present study was intended to evaluate the efficiency of gene transfection by Effectene® (Qiagen, Inc.), a lip...

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Bibliographic Details
Published in:Molecular reproduction and development 2005-10, Vol.72 (2), p.191-200
Main Authors: Lee, Sung-Lim, Ock, Sun-A, Yoo, Jae-Gyu, Kumar, B. Mohana, Choe, Sang-Yong, Rho, Gyu-Jin
Format: Article
Language:English
Subjects:
Animals
Apoptosis
Biological and medical sciences
Birth control
Cattle
Cell Cycle
Cell Nucleus - metabolism
cell types
Cells, Cultured
Chromosomes - genetics
Embryo, Mammalian - cytology
Embryo, Mammalian - embryology
Embryo, Mammalian - metabolism
Gene Expression
Genes, Reporter - genetics
Gynecology. Andrology. Obstetrics
Medical sciences
nuclear transfer
Nuclear Transfer Techniques
pEGFP-N1
Sterility. Assisted procreation
Time Factors
transfection
Transfection - methods
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Summary:The production of transgenic (TG) animals by somatic cell nuclear transfer (SCNT) has proven to be a more efficient method than other methods, such as gene injection or sperm mediation. The present study was intended to evaluate the efficiency of gene transfection by Effectene® (Qiagen, Inc.), a lipid‐based reagent compared to electroporation in fetal‐derived fibroblast cells (FFC), cumulus‐derived fibroblast cells (CFC), and adult ear skin‐derived fibroblast cells (AEFC). Parameters compared were factors such as chromosome abnormality, gene expression, and the incidence of apoptosis. Further, the TG embryos with transfected donor cells generated by electroporation or Effectene were compared to IVF and SCNT embryos in terms of rates of cleavage, blastocyst formation, and blastocyst cell number. Most of the cells (>80%) at confluence were at G0/G1 and considered to be suitable nuclear donors for cloning. Transfection with a plasmid containing the enhanced green fluorescent protein (pEGFP‐N1) gene into FFC did not increase the incidence of chromosomal abnormalities. The rates of apoptosis in different cell types transfected with pEGFP‐N1 were 3.3%–5.0%, and the values did not differ among groups. In addition, the rates of apoptosis in various cells between 5–7 and 20–22 cell passages did not differ. However, the efficiency of gene transfecton into FFC by Effectene reagent (14.2 ± 1.7) was significantly (P 
ISSN:1040-452X
1098-2795
DOI:10.1002/mrd.20297