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.
References
Balciunas et al (2006) Harnessing a high cargo-capacity transposon for genetic applications in vertebrates. PLoS Genet 2:e169
Bedell VM et al (2012) In vivo genome editing using a high-efficiency TALEN system. Nature 491:114–118
Clark J, Whitelaw B (2003) A future for transgenic livestock. Nat Rev Genet 4:825–833
Ivarie R (2006) Competitive bioreactor hens on the horizon. Trends Biotechnol 24:99–101
Ivics Z et al (2009) Transposon-mediated genome manipulation in vertebrates. Nat Methods 6:415–422
Kim YG, Cha J, Chandrasegaran S (1996) Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci USA 93:1156–1160
Lillico SG, McGrew MJ, Sherman A, Sang HM (2005) Transgenic chickens as bioreactors for protein-based drugs. Drug Discov Today 10:191–196
Lyall J et al (2011) Suppression of avian influenza transmission in genetically modified chickens. Science 331:223–226
Macdonald J et al (2012) Efficient genetic modification and germ-line transmission of primordial germ cells using piggyBac and Tol2 transposons. Proc Natl Acad Sci USA 109:1466–1472
McGrew MJ et al (2004) Efficient production of germline transgenic chickens using lentiviral vectors. EMBO Rep 5:728–733
Mittler R, Blumwald E (2010) Genetic engineering for modern agriculture: challenges and perspectives. Annu Rev Plant Biol 61:443–462
Modziac PA, Petitte JN (2004) Status of transgenic chicken models for developmental biology. Dev Dyn 229:414–421
Park TS, Han JY (2012) Piggybac transposition into primordial germ cells is an efficient tool for transgenesis in chickens. Proc Natl Acad Sci USA 109:9337–9341
Rashidi H, Sottile V (2009) The chick embryo: hatching a model for contemporary biomedical research. BioEssays 31:459–465
Sato Y et al (2007) Stable integration and conditional expression of electroporated transgenes in chicken embryos. Dev Biol 305:616–624
Smith CA, Sinclair AH (2001) Sex determination in the chicken embryo. J Exp Zool 290:691–699
Sun P et al (2012) Transgenic chimera quail production by microinjecting lentiviral vector into the blood vessel of the early embryo. Anim Sci J 83:291–298
van de Lavoir MC et al (2006) Germline transmission of genetically modified primordial germ cells. Nature 441:766–769
Vergara MN, Canto-Soler MV (2012) Rediscovering the chick embryo as a model to study retinal development. Neur Dev. doi:10.1186/1749-8104-7-22
Watanabe M et al (1994) Liposome-mediated DNA transfer into chicken primordial germ cells in vivo. Mol Rep Dev 38:268–274
Acknowledgments
We would like to thank the staff of the Small Animal Facility at AAHL and staff of the Werribee Animal Health Facility. Mini Tol2 transposon plasmids were kindly provided by Professor Stephen C. Ekker, Mayo Clinic Cancer Center, Rochester, MN, USA. Cvh antibody was kindly provided by Dr Craig Smith, Murdoch Children’s Research Institute, Melbourne, Australia. This work was funded by Malta Advanced Technologies (MAT), the technology arm of the EW Group.
Conflict of interest
None.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Tyack, S.G., Jenkins, K.A., O’Neil, T.E. et al. A new method for producing transgenic birds via direct in vivo transfection of primordial germ cells. Transgenic Res 22, 1257–1264 (2013). https://doi.org/10.1007/s11248-013-9727-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11248-013-9727-2