Efficient Gene Transfer into Chicken Gonads by Combining Transposons with Polyethylenimine

Abstract

Transposon mediated transfection is a promising, safe, and convenient way to generate transgenic chicken compared with virus-mediated technology and the in vitro modification of primordial germ cells (PGCs). To establish a simple method for in vivo transfection of chicken PGCs, we applied four different transposon systems (PB, SB, Tol2, and ZB) to investigate the gene transfer efficiency of chicken gonads via direct injection of a mixture of transposon and transposase plasmids and transfection reagent (polyethylenimine, PEI) into the subgerminal cavity of Hamburger and Hamilton stage 2-3 chick embryos. We also compared the effect of the amount of plasmids injected on the gene transfer efficiency of chicken gonads. We found that over 70% of the gonads were green fluorescent protein (GFP)-positive across all four transposon groups, and that the proportion of GFP-positive gonads was not significantly different between different transposons. Some GFP positive cells in gonads were confirmed as germ cells by co-labeling with the germ cell specific antibody. We also found that the proportions of GFP-positive gonads decreased significantly with a decrease of plasmid dose from 100 ng to 20 or 50 ng. Here we revealed that a combination of transposons with PEI is a simple and efficient method for gene transfer into chicken gonads and able to transfect PGCs in vivo that could be used for the production of transgenic chickens.

Cultivation and characterization of primordial germ cells from blue layer hybrids (Araucana crossbreeds) and generation of germline chimeric chickens

“…We found that the proportion of Venus-positive gonads in all three injected genotypes was about 92–100%. This is consistent with other studies using reporter carrying PGCs 41 , 42 . The Venus-transfected PGCs do not lose their ability to migrate from the peripheral blood into the developing gonads of the embryos.…”