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United States Department of Agriculture

Agricultural Research Service

Research Project: Development of New Technologies and Methods to Enhance the Utilization and Long-Term Storage of Poultry, Swine and Fish Gametes and Embryos

Location: Animal Biosciences and Biotechnology Laboratory

Title: Sperm-mediated transgenesis in chicken using a PiggyBac transposon system

Authors
item Quansah, Emmanuel -
item Long, Julie
item Donovan, David
item Becker, Stephen
item Telugu, Bhanu -
item Foster Frey, Juli
item Urwin, Nigel -

Submitted to: Poultry Science Association Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: March 20, 2014
Publication Date: N/A

Technical Abstract: Sperm-mediated transgenesis in chicken using a PiggyBac transposon system Emmanuel Quansah1,2, Julie Long2, David Donovan2, Stephen Becker2, Bhanu Telugu2, Juli Frey2, Nigel Urwin1 1,Charles Sturt University, Graham Center of Agricultural Innovation, Wagga Wagga. Australia and 2Beltsville Agricultural Research Center, ARS, USDA, Beltsville, Maryland. Towards development of transgenic chickens without the use of viral vectors, we are studying factors affecting sperm mediated gene transfer (SMGT) using a ‘PiggyBac’ vector. The plasmid pPBCAG-GFP contains 13 bp terminal inverted repeats flanking a GFP gene driven by the CAG promoter. A helper plasmid containing a piggyBac transposase gene is co-transformed to cause transposition of the GFP gene into TTAA chromosomal sites. Lipofectamine LTXTM (Invitrogen) (LPX) is a new generation transfection agent with low toxicity reportedly able to infect a wide range of cell types. Our experiments examined the effects of LPX and DNA on sperm that had been purified from seminal fluid using Accudenz gradient centrifugation. The impact of LPX alone (5, 10 or 15 µL) or in combination with the pPBCAG-GFP (5, 10 or 15 µg) on sperm viability and mobility at 25oC and 41oC (chicken body temperature) was studied by flow cytometry. Sperm viability was >90% with up to 3 h incubation for all treatments. Similarly, we demonstrated that LPX alone or in combination with pPBCAG-GFP had little impact on sperm viability (~90%) or mobility (~0.1) at 25oC for up to 3 h of incubation. A total of 2x108 sperm (purified from seminal plasma) transformed with 15 µL LPX and 15 µg pPBCAG-GFP DNA (1 h; 25oC) was inseminated into 13 White Leghorns hens. Additional hens were inseminated with un-transformed sperm (-ve control) or with LPX treated sperm (+ve control). Egg fertility at day 6 of incubation resulting from negative, positive control and pPBCAG-GFP transformed sperm was 17.4%, 7.7% and 14.3%, respectively. These results demonstrate that a combination of the plasmid and LPX did not negatively impact viability, mobility or fertility of chicken sperm; however the low fertility seen over all treatments and controls suggests purification of the sperm compromised fertility. Key words: Chicken, Lipofectamine LTX, piggyBac, Transformation, Viability

Last Modified: 11/22/2014
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