Protamine sulfate protects exogenous DNA against nuclease degradation but is unable to improve the efficiency of bovine sperm mediated transgenesis.
The present study investigated whether protamine sulfate can be used to improve the efficiency of bovine sperm mediated transgenesis (SMT) by protecting the plasmid pCX-EGFP against nuclease activity. A high proportion (31%) of bovine spermatozoa transfected with the plasmid pCX-EGFP maintain their motility. Using an in vitro assay, protamine sulfate protected the plasmid against degradation by DNase I. However, upon transfecting spermatozoa, the plasmid remained intact regardless of whether it was complexed to protamine sulfate. When in vitro fertilisation (IVF) was undertaken using transfected sperm, 14.6 and 10.2% of blastocysts derived from pCX-EGFP only and pCX-EGFP-protamine transfected sperm, respectively, were PCR positive for the plasmid. In conclusion, using spermatozoa transfected with either pCX-EGFP or pCX-EGFP-protamine complexes, produced PCR positive blastocysts after SMT. However, the use of protamine sulfate does not improve the efficiency of SMT suggesting that factors other than nuclease activity could be limiting.
Alderson
Jon
J
Reproductive Technologies Group, AgResearch Ltd, Ruakura Research Centre, East Street, Private Bag 3123, Hamilton, New Zealand. jon.alderson@agresearch.co.nz
Wilson
Ben
B
Laible
Goetz
G
Pfeffer
Peter
P
L'Huillier
Phil
P
D000818:Animals; D030801:Animals, Genetically Modified; D002417:Cattle / Q000235:genetics / Q000502:physiology*; D004247:DNA / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D046149:Embryo Culture Techniques / Q000662:veterinary; D005307:Fertilization in Vitro / Q000662:veterinary; D005456:Fluorescent Dyes; D018014:Gene Transfer Techniques / Q000662:veterinary; D008297:Male; D010957:Plasmids / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary; D011479:Protamines / Q000494:pharmacology*; D020554:Sperm Injections, Intracytoplasmic / Q000662:veterinary; D013081:Sperm Motility; D013094:Spermatozoa; D014162:Transfection / Q000379:methods / Q000662:veterinary*
D005456:Fluorescent Dyes; D011479:Protamines; D004247:DNA; D003851:Deoxyribonucleases