|QUANSAH, EMMANUEL - Charles Stuart University|
|TELUGU, BHANU - University Of Maryland|
|Foster Frey, Juli|
|URWIN, NIGEL - Charles Stuart University|
Submitted to: BARC Poster Day
Publication Type: Abstract Only
Publication Acceptance Date: 4/11/2014
Publication Date: 4/11/2014
Citation: Quansah, E., Long, J.A., Donovan, D.M., Becker, S.C., Telugu, B., Foster Frey, J.A., Urwin, N. 2014. Sperm-mediated transgenesis in chicken using a PiggyBac transposon system. BARC Poster Day. BARC Poster Day.
Technical Abstract: Towards development of transgenic chickens without the use of viral vectors, factors affecting sperm mediated gene transfer (SMGT) using a piggyBac vector are being studied. The piggyBac pPBCAG-LacZ contains 13bp terminal inverted repeats flanking a LacZ gene driven by the CAG promoter. A helper plasmid containing a piggyBac transposase gene is co-transformed to cause transposition of the LacZ 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 either 25oC or 41oC (chicken body temperature) for up to 3 h incubation was evaluated by flow cytometry and the sperm mobility assay. Sperm viability was >90% at all time points for all treatments at 41oC. Similarly, 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. Artificial insemination was conducted with transformed semen (15 µL LPX and 15 µg pPBCAG-LacZ DNA for 1 h at 25oC) using three different treatments: 1) seminal fluid (SF) removal by centrifugation at 600g for 10 min (T1); 2) removal of SF by Accudenz centrifugation at 1250g for 30 min (T2); and 3) no SF removal (T3). Non-transformed sperm for each treatment were used as controls. Ten and five White Leghorn hens were used for each DNA-treated and control group, respectively. Each hen received 2X108 sperm. Fertility for T1 controls were 38.9, 66.7 and 45.5% for week 1, 2 and 3, respectively. T2 controls resulted in fertilities of 46.4, 52.2 and 16.6% for the first 3 weeks after insemination. For T3, fertilities were 56.3, 88.9 and 53.3% for week 1, 2 and 3, respectively. Fertilities for first, second and third weeks of incubation resulting from T1 DNA were 46.9, 53.5 and 15.6%, respectively. T2 DNA resulted in 48.6, 48.5 and 21.7% whilst T3 DNA was 54.8, 65.4 and 39.0% for week 1, 2 and 3, respectively. These results demonstrate that a combination of the plasmid and LPX did not negatively impact viability, mobility or fertility of chicken sperm. The method of seminal plasma removal as well as DNA-treatment had a reducing effect on fertility. None of embryos resulting from the DNA treatments stained positive for ß-galactosidase.