Improved transfection efficiency of chicken gonadal primordial germ cells for the production of transgenic poultry.
Electroporation is a common method of DNA transfection for many types of eukaryotic cells, but has not been attempted in avian primordial germ cells (PGCs). DNA uptake in chicken primordial germ cells (PGCs) was tested using electroporation with and without dimethyl sulfoxide (DMSO). Gonadal tissue and chicken embryonic fibroblasts (CEFs) were isolated from 6-day-old embryos (stage 29), transfected with pCMV beta carrying the bacterial lacZ gene, and cultured for 24 h. Gonadal primordial germ cells (gPGCs) were purified from culture using a Ficoll gradient. The addition of DMSO significantly increased the transfection efficiency of gPGCs but had no effect on chicken embryonic fibroblasts. Electroporation of gPGCs resulted in an 80% transfection efficiency compared with about 17% observed with liposomes. Approximately 200 transfected gPGCs were injected into 2.5-day-old (stage 17) recipient embryos and the eggs were incubated for an additional 3.5 days, 7.5 days or to hatching. The exogenous gene was detectable in 100%, 67% and 41% of the 6-day-old (stage 29), 10-day-old (stage 36) recipient embryos and hatched chicks gonads, respectively. PCR analysis of DNA from the hatched chicks showed that exogenous lacZ DNA was detected only in the gonad and not the liver and heart. These results indicated that electroporation was a suitable means of transfecting avian gPCGs for the goal of producing transgenic poultry.
Hong
Y H
YH
Department of Animal Science and Technology, College of Agriculture and Life Sciences, Seoul National University, Suweon, Korea.
Moon
Y K
YK
Jeong
D K
DK
Han
J Y
JY
D000822:Animal Husbandry; D000818:Animals; D030801:Animals, Genetically Modified; D002642:Chick Embryo; D002645:Chickens / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004121:Dimethyl Sulfoxide; D018274:Electroporation / Q000379:methods*; D015870:Gene Expression; D005821:Genetic Techniques; D005854:Germ Cells / Q000378:metabolism*; D007763:Lac Operon; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D001616:beta-Galactosidase; D004121:Dimethyl Sulfoxide