|dc.description.abstract||The chicken is a well-established model system for studying vertebrate embryogenesis, but creating transgenic birds has proven difficult. Viral infections have been predominantly used to insert transgenes and have been moderately successful, however, the rate of germ line infection is low and the virus is not easily manipulated in the laboratory. To increase efficiency and ease of production we are using piggyBac, a transposable element (TE) system, paired with an in-vivo transfection reagent, JetPEI, to generate transgenic chicks.
The piggyBac system utilizes a transposase enzyme, which recognizes a specific DNA sequence called a transposon. The enzyme excises the transposon from its original location in the DNA and inserts it into a new genomic location. The transposon used in our studies contains a constitutively expressed green fluorescent protein (GFP) gene for tracking insertion by fluorescent microscopy. The TE system is delivered to cells using JetPEI, an in-vivo transfection reagent. JetPEI forms a capsule around DNA, which is endocytosed by cells. Once inside, the capsules rupture and TE DNA is released into the cell to integrate into the genome. The current question with this system is two-fold. First, is JetPEI able to transfect developing chick cells, namely germ cells and, second, will piggyBac efficiently integrate into the chick genome.
To answer this question we prepared a mixture of JetPEI with the piggyBac system. This was injected into Stage X white leghorn embryos which were incubated to hatch. Hatched chicks of differing ages were euthanized to evaluate GFP expression. Imaging showed GFP expression in multiple tissue types from all three germ layers. 6/18 males expressed GFP in the testes, which co-localized with positive staining from germ cell antibodies showing that JetPEI will transfect germ cells. Stable expression of GFP in multiple tissue types including testes from different age chicks indicates that piggyBac has integrated into the genome and is actively expressing the transgene. The efficiency and ease of manipulation of piggyBac in combination with JetPEI make this a powerful system for creating transgenic chicks.||