CpG methylation modulates tissue-specific expression of a transgene in chickens.
The use of genetically modified germ cells is an ideal system to induce transgenesis in birds; the primordial germ cell (PGC) is the most promising candidate for this system. In the present study, we confirmed the practical application of this system using lentivirus-transduced chicken gonadal PGCs (gPGCs). Embryonic gonads were collected from 5.5-d old Korean Oge chickens (black feathers). The gPGC population was enriched (magnetic-activated cell sorting technique) and then they were transduced with a lentiviral vector expressing enhanced green fluorescent protein (eGFP), under the control of the Rous sarcoma virus (RSV) promoter. Subsequently, the eGFP-transduced PGCs were transplanted into blood vessels of 2.5-d-old embryonic White Leghorn (white feathers). Among 21 germline chimeric chickens, one male produced transgenic offspring (G(1) generation), as demonstrated by testcross and genetic analysis. A homozygous line was produced and maintained through the G(3) generation. Based on serum biochemistry, there were no significant physiological differences between G(3) homozygotes and non-transgenic chickens. However, since eGFP transgene expression in G(3) chickens varied among tissues, it was further characterized by Western blotting and ELISA. Furthermore, there were indications that DNA methylation may have affected tissue-specific expression of transgenes in chickens. In conclusion, the PGC-mediated approach used may be an efficient tool for avian transgenesis, and transgenic chickens could provide a useful model for investigating regulation of gene expression.
Park
Sang Hyun
SH
Avicore Biotechnology Institute, Optifarm Solution Inc., Hanlim Human Tower #707, Gyeonggi-do, Korea.
Kim
Jin Nam
JN
Park
Tae Sub
TS
Lee
Seon Duk
SD
Kim
Tae Hyun
TH
Han
Beom Ku
BK
Han
Jae Yong
JY
D000818:Animals; D030801:Animals, Genetically Modified / Q000097:blood / Q000378:metabolism; D015153:Blotting, Western; D002642:Chick Embryo / Q000166:cytology*; D002645:Chickens / Q000097:blood / Q000235:genetics*; D002678:Chimera; D018899:CpG Islands; D019175:DNA Methylation; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D005822:Genetic Vectors; D005854:Germ Cells / Q000637:transplantation*; D049452:Green Fluorescent Proteins / Q000032:analysis; D016086:Lentivirus / Q000235:genetics; D014161:Transduction, Genetic / Q000662:veterinary; D019076:Transgenes
D049452:Green Fluorescent Proteins