title: Establishment and characterization of fetal fibroblast cell lines for generating human lysozyme transgenic goats by somatic cell nuclear transfer.
abstract: This study was performed to qualify goat fetal fibroblast (GFF) cell lines for genetic modification and somatic cell nuclear transfer (SCNT) to produce human lysozyme (hLYZ) transgenic goats. Nine GFF cell lines were established from different fetuses, and the proliferative lifespan and chromosomal stability were analyzed. The results suggested that cell lines with a longer lifespan had stable chromosomes compared with those of cells lines with a shorter lifespan. According to the proliferative lifespan, we divided GFF cell lines into two groups: cell lines with a long lifespan (GFF1/2/7/8/9; group L) and cell lines with a short lifespan (GFF3/4/5/6; group S). Next, a hLYZ expression vector was introduced into these cell lines by electroporation. The efficiencies of colony formation, expansion in culture, and the quality of transgenic clonal cell lines were significant higher in group L than those in group S. The mean fusion rate and blastocyst rate in group L were higher than those in group S (80.3 ± 1.7 vs. 65.1 ± 4.2 % and 19.5 ± 0.6 vs. 15.1 ± 1.1 %, respectively, P < 0.05). After transferring cloned embryos into the oviducts of recipient goats, three live kids were born. PCR and Southern blot analyses confirmed integration of the transgene in cloned goats. In conclusion, the lifespan of GFF cell lines has a major effect on the efficiency to produce transgenic cloned goats. Therefore, the proliferative lifespan of primary cells may be used as a criterion to characterize the quality of cell lines for genetic modification and SCNT.
authors: College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China.
authors: Jun
authors: J
authors: Liu
authors: Yan
authors: Y
authors: Luo
authors: Liming
authors: L
authors: Zheng
authors: Qingqing
authors: Q
authors: Liu
authors: Zhongcai
authors: Z
authors: Yang
authors: Yongsheng
authors: Y
authors: Wang
authors: Jianmin
authors: J
authors: Su
authors: Fusheng
authors: F
authors: Quan
authors: Yong
authors: Y
authors: Zhang
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D015139:Blotting, Southern; D002460:Cell Line; D002470:Cell Survival / Q000235:genetics; D043171:Chromosomal Instability / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D018274:Electroporation; D004624:Embryo Transfer; D005347:Fibroblasts / Q000166:cytology*; D005822:Genetic Vectors / Q000235:genetics; D006041:Goats / Q000235:genetics*; D006801:Humans; D009113:Muramidase / Q000235:genetics*; D053652:Nuclear Transfer Techniques; D016133:Polymerase Chain Reaction; D014162:Transfection
chemical_list: D017931:DNA Primers; D009113:Muramidase