A new method for producing transgenic birds via direct in vivo transfection of primordial germ cells

Abstract

Traditional methods of avian transgenesis involve complex manipulations involving either retroviral infection of blastoderms or the ex vivo manipulation of primordial germ cells (PGCs) followed by injection of the cells back into a recipient embryo. Unlike in mammalian systems, avian embryonic PGCs undergo a migration through the vasculature on their path to the gonad where they become the sperm or ova producing cells. In a development which simplifies the procedure of creating transgenic chickens we have shown that PGCs are directly transfectable in vivo using commonly available transfection reagents. We used Lipofectamine 2000 complexed with Tol2 transposon and transposase plasmids to stably transform PGCs in vivo generating transgenic offspring that express a reporter gene carried in the transposon. The process has been shown to be highly effective and as robust as the other methods used to create germ-line transgenic chickens while substantially reducing time, infrastructure and reagents required. The method described here defines a simple direct approach for transgenic chicken production, allowing researchers without extensive PGC culturing facilities or skills with retroviruses to produce transgenic chickens for wide-ranging applications in research, biotechnology and agriculture.

Efficient Gene Transfer into Chicken Gonads by Combining Transposons with Polyethylenimine

“…To find the most efficient transposon, we compared the transfection efficiency of PGCs across four transposons by screening the GFP-positive gonads. All of these transposons were highly efficient, and no significant difference was observed in the proportion of GFP-positive gonads, which also suggests that all four of these transposons are applicable in chicken transgenesis and that their efficiency is consistent with that previously reported (Tyack et al, 2013). We also found that combining PEI with a high dose (100 ng) of plasmid can generate a high proportion of GFP-positive gonads, while combining PEI with a low dose (20 or 50 ng) of plasmid can generate a low proportion of GFP-positive gonads, but with a limited increase in the hatching rates and viability of embryos, which indicates that the hatching rates and viability of embryos may be affected mainly by the physical injury of the injection, not the dose of plasmid or transfection reagent.…”

“…10;2016 Although the transfection of PGCs can be achieved by injecting plasmid DNA and liposome complexes into the bloodstream of stage HH 14 embryos (Watanabe et al, 1994), this method is inefficient and unstable without a transposon medium. Combining transposons with transfection reagents has been shown to be effective for chimera production and germline transgenesis by direct injection into the vasculature of developing chick embryos or stage X embryos (Jordan et al, 2014;Tyack et al, 2013). The combination of Tol2 transposon with Lipofectamine 2000 has been confirmed to transfect the PGCs efficiently in vivo by direct injection into the vasculature of developing chick embryos and to generate stable germ-line transgenic male chickens who passed the transgene onto their offspring (Tyack et al, 2013).…”

“…Combining transposons with transfection reagents has been shown to be effective for chimera production and germline transgenesis by direct injection into the vasculature of developing chick embryos or stage X embryos (Jordan et al, 2014;Tyack et al, 2013). The combination of Tol2 transposon with Lipofectamine 2000 has been confirmed to transfect the PGCs efficiently in vivo by direct injection into the vasculature of developing chick embryos and to generate stable germ-line transgenic male chickens who passed the transgene onto their offspring (Tyack et al, 2013). That study (Tyack et al, 2013) revealed that all gonads sampled at ED 14 were GFP positive (10 out of 10 embryos), five out of the 11 F0 male semen were transgene positive, five out of the 419 offspring from the F0 individuals with transgene positive semen were GFP positive, and the transgene rate of their offspring was about 1.5%, which is substantially lower than that using lentiviral vectors (4-45%) (McGrew et al, 2004).…”

Welcome to the CRISPR zoo

“…Researchers can therefore remove PGCs, edit them in the lab and then return them to the developing bird. The CSIRO team has even developed a method to insert CRISPR components directly into the bloodstream so that they can edit PGCs there 4 . The researchers also plan to produce chickens with components required for CRISPR integrated directly into their genomes -what they call CRISPi chickens.…”

Isolation, Cultivation and Characterization of Quail Primordial Germ Cells

“…The use of this type of cells in selection and biotechnological programs opens wide opportunities for directed genome modification and the reconstruction of valuable breeds and lines preserved in cryobank conditions [6][7][8][9][10][11][12]. Peculiarities of biology of agricultural poultry allow us to consider PGC as a promising genetic material.…”