Cell cycle distribution, cellular viability and mRNA expression of hGCase-gene-transfected cells in dairy goat.
Nuclear transfer using transgenic donor cells is an efficient way of generating transgenic goats, wherein the preparation of competent transgenic donor cells is the pivotal upstream step. We have measured the efficiency of transfection with a plasmid containing hGCase (human lysosomal acid beta-glucosidase) gene into goat FFC (fetal-derived fibroblast cells), MEC (mammary epithelial cells) and AEFC (adult ear skin-derived fibroblast cells), and the characteristics of cell cycle, apoptosis and chromosome abnormalities after transfection. The expression of genes involved in imprinting [IGF2 (insulin-like growth factor 2), IGF2R (IGF2 receptor)], apoptosis (Bax), stress (heat-shock protein, Hsp70.1), cellular connections [Cx43 (connexin 43)] and DNA methylation [DNMT1 (DNA methyltransferase 1)] in transgenic fetal cells has been investigated. The hGCase transgene was successfully detected in the transfected cell lines, and chromosomal stability remained similar in FFC and transgenic FFC (70.9 compared with 66.8%), whereas a smaller percentage (P<0.05) of cells at G(0)/G(1) in the transgenic FFC, MEC and AEFC (T-FFC, T-MEC and T-AEFC), and higher percentage (P<0.05) of apoptotic cells in T-FFC than the non-transfected controls were detected by flow cytometric analysis. Among the genes tested, the relative expressions of IGF2, IGF2R and transcripts of Cx43 were significantly higher (P<0.05) in T-FFC compared with non-transfected FFC. These novel findings on gene expression in transgenic fetal cells may have certain implications in the biopharming industry and in our understanding the low efficiency of transgenic cloning.
Zhang
Yan-Li
YL
Center of Embryo Engineering and Technology, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China.
Wan
Yong-Jie
YJ
Wang
Zi-Yu
ZY
Qi
Wei-Wei
WW
Zhou
Zheng-Rong
ZR
Huang
Rong
R
Wang
Feng
F
D000818:Animals; D017209:Apoptosis; D002455:Cell Division; D002461:Cell Line, Transformed; D002470:Cell Survival; D043171:Chromosomal Instability; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D005962:Glucosylceramidase / Q000235:genetics* / Q000378:metabolism; D006041:Goats / Q000235:genetics; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000235:genetics / Q000378:metabolism; D007399:Interphase; D012333:RNA, Messenger / Q000378:metabolism; D017527:Receptor, IGF Type 2 / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D019076:Transgenes
D018031:Connexin 43; D018840:HSP70 Heat-Shock Proteins; D012333:RNA, Messenger; D017527:Receptor, IGF Type 2; C081884:heat-shock protein 70.1; D007335:Insulin-Like Growth Factor II; D004248:DNA (Cytosine-5-)-Methyltransferases; D005962:Glucosylceramidase