Protamine sulfate protects exogenous DNA against nuclease degradation but is unable to improve the efficiency of bovine sperm mediated transgenesis
| Autor: | Götz Laible, Peter L. Pfeffer, Jon Alderson, Ben Wilson, Phil L'Huillier |
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| Rok vydání: | 2004 |
| Předmět: |
Male
Protamine sulfate Fertilization in Vitro Transfection Polymerase Chain Reaction Animals Genetically Modified Embryo Culture Techniques Endocrinology Plasmid Food Animals medicine Animals Protamines Sperm Injections Intracytoplasmic reproductive and urinary physiology Fluorescent Dyes Nuclease Deoxyribonucleases biology urogenital system fungi Gene Transfer Techniques General Medicine DNA Molecular biology Sperm Spermatozoa In vitro Transgenesis embryonic structures biology.protein Sperm Motility Animal Science and Zoology Exogenous DNA Cattle medicine.drug Plasmids |
| Zdroj: | Animal reproduction science. 91(1-2) |
| ISSN: | 0378-4320 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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